Cysteine engineered anti-tenb2 antibodies and antibody drug conjugates

ABSTRACT

Cysteine engineered anti-TENB2 antibodies are engineered by replacing one or more amino acids of a parent anti-TENB2 antibody with non cross-linked, reactive cysteine amino acids. Methods of design, preparation, screening, and selection of the cysteine engineered anti-TENB2 antibodies are provided. Cysteine engineered anti-TENB2 antibodies (Ab) are conjugated with one or more drug moieties (D) through a linker (L) to form cysteine engineered anti-TENB2 antibody-drug conjugates having Formula I: 
       Ab-(L-D) p   I
         where p is 1 to 4. Diagnostic and therapeutic uses for cysteine engineered antibody drug compounds and compositions are disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional application of U.S. application Ser.No. 12/288,181, filed Oct. 16, 2008, which is a non-provisionalapplication filed under 37 CFR 1.53(b)(1), claiming priority under 35USC 119(e) to provisional application No. 60/981,411 filed Oct. 19,2007, the entire disclosures of which are incorporated herein byreference in their entirety.

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. The ASCII copy, created on Nov. 18, 2014, istitled “GNE0311 US” and is 28,672 bytes in size.

FIELD OF THE INVENTION

The invention relates generally to antibodies engineered with reactivecysteine residues and more specifically to antibodies with therapeuticor diagnostic applications. The cysteine engineered antibodies may beconjugated with chemotherapeutic drugs, toxins, affinity ligands such asbiotin, and detection labels such as fluorophores. The invention alsorelates to methods of using antibodies and antibody-drug conjugatecompounds for in vitro, in situ, and in vivo diagnosis or treatment ofmammalian cells, or associated pathological conditions.

BACKGROUND OF THE INVENTION

Antibody therapy has been established for the targeted treatment ofpatients with cancer, immunological and angiogenic disorders.Transmembrane or otherwise tumor-associated polypeptides specificallyexpressed on the surface of cancer cells as compared to normal,non-cancerous cell(s) have been identified as cellular targets forcancer diagnosis and therapy with antibodies. Identification of suchtumor-associated cell surface antigen polypeptides, i.e. tumorassociated antigens (TAA), allows specific targeting of cancer cells fordestruction via antibody-based therapies.

The use of antibody-drug conjugates (ADC), i.e. immunoconjugates, forthe local delivery of cytotoxic or cytostatic agents, i.e. drugs to killor inhibit tumor cells in the treatment of cancer (Lambert, J. (2005)Curr. Opinion in Pharmacology 5:543-549; Wu et al (2005) NatureBiotechnology 23(9): 1137-1146; Payne, G. (2003) Cancer Cell 3:207-212;Syrigos and Epenetos (1999) Anticancer Research 19:605-614;Niculescu-Duvaz and Springer (1997) Adv. Drug Del. Rev. 26:151-172; U.S.Pat. No. 4,975,278) allows targeted delivery of the drug moiety totumors, and intracellular accumulation therein, where systemicadministration of these unconjugated drug agents may result inunacceptable levels of toxicity to normal cells as well as the tumorcells sought to be eliminated (Baldwin et al (1986) Lancet pp. (Mar. 15,1986):603-05; Thorpe, (1985) “Antibody Carriers Of Cytotoxic Agents InCancer Therapy: A Review,” in Monoclonal Antibodies '84: Biological AndClinical Applications, A. Pinchera et al (ed.s), pp. 475-506). Effortsto improve the therapeutic index, i.e. maximal efficacy and minimaltoxicity of ADC have focused on the selectivity of polyclonal (Rowlandet al (1986) Cancer Immunol. Immunother., 21:183-87) and monoclonalantibodies (mAbs) as well as drug-linking and drug-releasing properties(Lambert, J. (2005) Curr. Opinion in Pharmacology 5:543-549). Drugmoieties used in antibody drug conjugates include bacterial proteintoxins such as diphtheria toxin, plant protein toxins such as ricin,small molecules such as auristatins, geldanamycin (Mandler et al (2000)J. of the Nat. Cancer Inst. 92(19):1573-1581; Mandler et al (2000)Bioorganic & Med. Chem. Letters 10:1025-1028; Mandler et al (2002)Bioconjugate Chem. 13:786-791), maytansinoids (EP 1391213; Liu et al(1996) Proc. Natl. Acad. Sci. USA 93:8618-8623), calicheamicin (Lode etal (1998) Cancer Res. 58:2928; Hinman et al (1993) Cancer Res.53:3336-3342), daunomycin, doxorubicin, methotrexate, and vindesine(Rowland et al (1986) supra). The drug moieties may affect cytotoxic andcytostatic mechanisms including tubulin binding, DNA binding, ortopoisomerase inhibition. Some cytotoxic drugs tend to be inactive orless active when conjugated to large antibodies or protein receptorligands.

The auristatin peptides, auristatin E (AE) and monomethylauristatin(MMAE), synthetic analogs of dolastatin (WO 02/088172), have beenconjugated as drug moieties to: (i) chimeric monoclonal antibodies cBR96(specific to Lewis Y on carcinomas); (ii) cAC 10 which is specific toCD30 on hematological malignancies (Klussman, et al (2004), BioconjugateChemistry 15(4):765-773; Doronina et al (2003) Nature Biotechnology21(7):778-784; Francisco et al (2003) Blood 102(4):1458-1465; US2004/0018194; (iii) anti-CD20 antibodies such as rituxan (WO 04/032828)for the treatment of CD20-expressing cancers and immune disorders; (iv)anti-EphB2R antibody 2H9 for treatment of colorectal cancer (Mao et al(2004) Cancer Research 64(3):781-788); (v) E-selectin antibody (Bhaskaret al (2003) Cancer Res. 63:6387-6394); (vi) trastuzumab (HERCEPTIN®, US2005/0238649), and (vi) anti-CD30 antibodies (WO 03/043583). Variants ofauristatin E are disclosed in U.S. Pat. No. 5,767,237 and U.S. Pat. No.6,124,431. Monomethyl auristatin E conjugated to monoclonal antibodiesare disclosed in Senter et al, Proceedings of the American Associationfor Cancer Research, Volume 45, Abstract Number 623, presented Mar. 28,2004. Auristatin analogs MMAE and MMAF have been conjugated to variousantibodies (US 2005/0238649).

Conventional means of attaching, i.e. linking through covalent bonds, adrug moiety to an antibody generally leads to a heterogeneous mixture ofmolecules where the drug moieties are attached at a number of sites onthe antibody. For example, cytotoxic drugs have typically beenconjugated to antibodies through the often-numerous lysine residues ofan antibody, generating a heterogeneous antibody-drug conjugate mixture.Depending on reaction conditions, the heterogeneous mixture typicallycontains a distribution of antibodies with from 0 to about 8, or more,attached drug moieties. In addition, within each subgroup of conjugateswith a particular integer ratio of drug moieties to antibody, is apotentially heterogeneous mixture where the drug moiety is attached atvarious sites on the antibody. Analytical and preparative methods may beinadequate to separate and characterize the antibody-drug conjugatespecies molecules within the heterogeneous mixture resulting from aconjugation reaction. Antibodies are large, complex and structurallydiverse biomolecules, often with many reactive functional groups. Theirreactivities with linker reagents and drug-linker intermediates aredependent on factors such as pH, concentration, salt concentration, andco-solvents. Furthermore, the multistep conjugation process may benonreproducible due to difficulties in controlling the reactionconditions and characterizing reactants and intermediates.

Cysteine thiols are reactive at neutral pH, unlike most amines which areprotonated and less nucleophilic near pH 7. Since free thiol (RSH,sulfhydryl) groups are relatively reactive, proteins with cysteineresidues often exist in their oxidized form as disulfide-linkedoligomers or have internally bridged disulfide groups. Extracellularproteins generally do not have free thiols (Garman, 1997,Non-Radioactive Labelling: A Practical Approach, Academic Press, London,at page 55). Antibody cysteine thiol groups are generally more reactive,i.e. more nucleophilic, towards electrophilic conjugation reagents thanantibody amine or hydroxyl groups. Cysteine residues have beenintroduced into proteins by genetic engineering techniques to formcovalent attachments to ligands or to form new intramolecular disulfidebonds (Better et al (1994) J. Biol. Chem. 13:9644-9650; Bernhard et al(1994) Bioconjugate Chem. 5:126-132; Greenwood et al (1994) TherapeuticImmunology 1:247-255; Tu et al (1999) Proc. Natl. Acad. Sci USA96:4862-4867; Kanno et al (2000) J. of Biotechnology, 76:207-214; Chmuraet al (2001) Proc. Nat. Acad. Sci. USA 98(15):8480-8484; U.S. Pat. No.6,248,564). However, engineering in cysteine thiol groups by themutation of various amino acid residues of a protein to cysteine aminoacids is potentially problematic, particularly in the case of unpaired(free Cys) residues or those which are relatively accessible forreaction or oxidation. In concentrated solutions of the protein, whetherin the periplasm of E. coli, culture supernatants, or partially orcompletely purified protein, unpaired Cys residues on the surface of theprotein can pair and oxidize to form intermolecular disulfides, andhence protein dimers or multimers. Disulfide dimer formation renders thenew Cys unreactive for conjugation to a drug, ligand, or other label.Furthermore, if the protein oxidatively forms an intramoleculardisulfide bond between the newly engineered Cys and an existing Cysresidue, both Cys thiol groups are unavailable for active siteparticipation and interactions. Furthermore, the protein may be renderedinactive or non-specific, by misfolding or loss of tertiary structure(Zhang et al (2002) Anal. Biochem. 311:1-9).

Cysteine-engineered antibodies have been designed as FAB antibodyfragments (thioFab) and expressed as full-length, IgG monoclonal(thioMab) antibodies (US 2007/0092940, the contents of which areincorporated by reference). ThioFab and ThioMab antibodies have beenconjugated through linkers at the newly introduced cysteine thiols withthiol-reactive linker reagents and drug-linker reagents to prepareantibody drug conjugates (Thio ADC).

TENB2 is a tumor associated antigen polypeptide (also known as PR1), andthe TENB2 protein contains 2 follistatin-like domains and a conservedEGF-like domain. The gene encoding the protein was first characterizedfrom a human brain cDNA library (see Uchida, et al. (1999) Biochem.Biophys. Res. Commun. 266:593-602), and later isolated from a humanfetal brain cDNA library (see Horie, et al. (2000) Genomics 67:146-152).See also, e.g., Online Mendelian Inheritance in Man, number 605734;Unigene Cluster Hs.22791; LocusLink 23671; and other linked sites. TENB2has been referred to as PR1, tomoregulin, TR, hyperplastic polyposisgene 1, HPP1, and TMEFF2. It's nucleic acid sequence can be identifiedby ATCC Accession Nos. AF264150, AB004064, AB017269, and AF179274; andit's amino acid sequence can be identified by ATCC Accession Nos.AAF91397, BAA90820, BAA87897, and AAD55776. TENB2's UniGene Clusteridentification number is hs.22791, Locuslink identification number is23671, and OMIM identification number is 605734.

The gene has also been implicated in certain cancerous conditions.Young, et al. (2001) Proc. Nat'l Acad. Sci. USA 98:265-270 reportedexpression in colorectal polyps. Glynne-Jones, et al. (2001) Int. J.Cancer 94:178-184 reported it as a marker for prostate cancer.

Due to its overexpression in certain human tumors, the TENB2 polypeptideand the nucleic acid encoding that polypeptide are targets forquantitative and qualitative comparisons among various mammalian tissuesamples. The unique expression profiles of TENB2 polypeptide, and thenucleic acid encoding that polypeptide, can be exploited for thediagnosis and therapeutic treatment of certain types of cancerous tumorsin mammals.

Recently, certain anti-TENB2 antibodies, including anti-TMEFF2 antibody#19, were disclosed and shown to be internalized and useful for thetreatment of proliferative conditions of the prostate, including, e.g.,benign prostate hyperplasia and prostate cancer (PCT/US03/07209; U.S.Ser. No. 10/383,447, filed Mar. 7, 2003, now U.S. Pat. No. 7,288,248;Vinay et al., “Antibodies Against Cancer Antigen TMEFF2 and UsesThereof” the contents of which are incorporated by reference).

SUMMARY

In one aspect, the invention includes a cysteine engineered anti-TENB2antibody comprising one or more free cysteine amino acids and a sequenceselected from SEQ ID NOS:8-23. The cysteine engineered anti-TENB2antibody may bind to a TENB2 polypeptide. Tumor-associated antigens(TAA) such as TENB2 polypeptides can be prepared for use in generatingcysteine engineered antibodies using methods and information which arewell known in the art, and for example in PCT/US03/07209 (U.S. Pat. No.7,288,248). The cysteine engineered anti-TENB2 antibody may be preparedby a process comprising replacing one or more amino acid residues of aparent anti-TENB2 antibody by cysteine.

The one or more free cysteine amino acid residues of the cysteineengineered anti-TENB2 antibody are located in a light chain or a heavychain.

In one aspect, the invention includes a method of determining thepresence of a TENB2 protein in a sample suspected of containing saidprotein, said method comprising exposing said sample to a cysteineengineered anti-TENB2 antibody and determining binding of said antibodyto said TENB2 protein in said sample, wherein binding of the antibody tosaid protein is indicative of the presence of said protein in saidsample.

Cysteine engineered anti-TENB2 antibodies may be used as nakedantibodies (unconjugated to a drug or label moiety) or as antibody-drugconjugates (ADC). The cysteine engineered anti-TENB2 antibody may becovalently attached to an auristatin drug moiety whereby an antibodydrug conjugate is formed. The antibody-drug conjugate may comprising acysteine engineered anti-TENB2 antibody (Ab), and an auristatin drugmoiety (D) wherein the cysteine engineered anti-TENB2 antibody isattached through one or more free cysteine amino acids by a linkermoiety (L) to D; the compound having Formula I:

Ab-(L-D)_(p)  I

-   -   where p is 1, 2, 3, or 4. Auristatin drug moieties include MMAE        and MMAF.

An aspect of the invention is an assay for detecting cancer cellscomprising: (a) exposing cells to an antibody-drug conjugate compound;and (b) determining the extent of binding of the antibody-drug conjugatecompound to the cells.

An aspect of the invention is a pharmaceutical formulation comprisingthe antibody drug conjugate, and a pharmaceutically acceptable diluent,carrier or excipient.

An aspect of the invention is a method of inhibiting cellularproliferation comprising treating mammalian tumor cells in a cellculture medium with an antibody-drug conjugate compound, wherebyproliferation of the tumor cells is inhibited.

An aspect of the invention is a method of treating cancer comprisingadministering to a patient the pharmaceutical formulation. The patientmay be administered a chemotherapeutic agent in combination with theantibody-drug conjugate compound.

An aspect of the invention is an article of manufacture comprising thepharmaceutical formulation, a container; and a package insert or labelindicating that the compound can be used to treat cancer characterizedby the overexpression of a TENB2 polypeptide.

An aspect of the invention is a method for making a Formula I antibodydrug conjugate compound comprising the steps of: (a) reacting anengineered cysteine group of the cysteine engineered antibody with alinker reagent to form antibody-linker intermediate Ab-L; and (b)reacting Ab-L with an activated drug moiety D; whereby the antibody-drugconjugate is formed; or comprising the steps of: (c) reacting anucleophilic group of a drug moiety with a linker reagent to formdrug-linker intermediate D-L; and (d) reacting D-L with an engineeredcysteine group of the cysteine engineered antibody; whereby theantibody-drug conjugate is formed.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the Heavy Chain sequence: SEQ ID NO: 1, and Light Chainsequence: SEQ ID NO:2 of humanized anti-TENB2 antibody, hu TMEFF2#19.

FIG. 2 shows the Heavy Chain sequence: SEQ ID NO:3, and Light Chainsequence: SEQ ID NO:2 of a humanized cysteine engineered anti-TENB2antibody, A121C thio hu TMEFF2#19. Signal sequence is not included inthe sequential numbering of anti-TENB2 antibody.

FIG. 3 shows alignment of humanized trastuzumab light chain (HuTMAb-LC,SEQ ID NO:4) and hu TMEFF2#19 light chain (SEQ ID NO:5). The numberingfollows the sequential numbering convention.

FIG. 4 shows alignment of humanized trastuzumab heavy chain (HuTMAb-HC,SEQ ID NO:6), and hu TMEFF2#19 heavy chain (SEQ ID NO:7). The numberingfollows the sequential numbering convention.

FIG. 5 shows depictions of cysteine engineered anti-TENB2 antibody drugconjugates (ADC) where a drug moiety is attached to an engineeredcysteine group in: the light chain (LC-ADC); the heavy chain (HC-ADC);and the Fc region (Fc-ADC).

FIG. 6 shows the steps of: (i) reducing cysteine disulfide adducts andinterchain and intrachain disulfides in a cysteine engineered anti-TENB2antibody (ThioMab); (ii) partially oxidizing, i.e. reoxidation to reforminterchain and intrachain disulfides; and (iii) conjugation of thereoxidized antibody with a drug-linker intermediate to form a cysteineengineered anti-TENB2 antibody drug conjugate (ADC).

FIG. 7 shows expression of TENB2 in cancer and normal human tissues:oligonucleotide microarray analysis was performed on RNA extracted from4841 human tissue samples. Each box in the plot provides signalintensity (average difference scaled to 100) for TENB2 for a sample ofthe indicated tissue. Green boxes are normal tissue, red boxes aretumors, and blue boxes represent other diseased tissues.

FIG. 8 shows TENB2 expression in human prostate tumors: Top and bottompanels are from human prostate explant models, PC3TENB2 medium stablecell line with vector control and prostate tumor, respectively.

FIG. 9 shows internalization of TENB2 monoclonal antibody (Mab) onPC3TENB2 Medium cell line and LuCaP 70 tumor.

FIGS. 10A and B shows FACS data on PC3 TENB2 Medium cells with thio(FIG. 10B) or conventional anti-TENB2 ADC treatment (FIG. 10A).

FIGS. 11A and B shows a cell killing assay on PC3 TENB2 Medium cellswith conventional anti-TENB2 (FIG. 11A) and thio-anti-TENB2 ADCs (FIG.11B).

FIG. 12 shows an efficacy study on PC3 TENB2 Medium cells usinganti-TENB2 and thio-anti-TENB2 ADCs (conjugated with vc-MMAE orMC-MMAF).

FIG. 13 shows a Western Blot with various LuCaP explant tumor tissuesusing humanized anti-TENB2 Ab (hu TMEFF2#19).

FIG. 14 shows xenograft experiments using human prostate cancer LuCaP70, 77 and 96.1.

FIG. 15 shows pharmacokinetic evaluation of rats using thio-anti-TENB2and conventional ADCs.

FIG. 16 shows a safety assessment on rats with anti-TENB2-vc-MMAE vs.MC-MMAF.

FIGS. 17A and B shows a safety assessment on cynomolgus monkeys withanti-TENB2-vc-MMAE vs. anti-TENB2-MC-MMAF.

FIGS. 18A and B shows a safety assessment on rats withthio-anti-TENB2-vc-MMAE vs. anti-TENB2-vc-MMAE.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Reference will now be made in detail to certain embodiments of theinvention, examples of which are illustrated in the accompanyingstructures and formulas. While the invention will be described inconjunction with the enumerated embodiments, it will be understood thatthey are not intended to limit the invention to those embodiments. Onthe contrary, the invention is intended to cover all alternatives,modifications, and equivalents, which may be included within the scopeof the present invention as defined by the claims.

One skilled in the art will recognize many methods and materials similaror equivalent to those described herein, which could be used in thepractice of the present invention. The present invention is in no waylimited to the methods and materials described.

DEFINITIONS

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs, and are consistent with:Singleton et al (1994) Dictionary of Microbiology and Molecular Biology,2nd Ed., J. Wiley & Sons, New York, N.Y.; and Janeway, C., Travers, P.,Walport, M., Shlomchik (2001) Immunobiology, 5th Ed., GarlandPublishing, New York.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies, polyclonal antibodies,dimers, multimers, multispecific antibodies (e.g., bispecificantibodies), and antibody fragments, so long as they exhibit the desiredbiological activity (Miller et al (2003) Jour. of Immunology170:4854-4861). Antibodies may be murine, human, humanized, chimeric, orderived from other species. An antibody is a protein that is capable ofrecognizing and binding to a specific antigen (Janeway, C., Travers, P.,Walport, M., Shlomchik (2001) Immuno Biology, 5th Ed., GarlandPublishing, New York). A target antigen generally has numerous bindingsites, also called epitopes, recognized by CDRs on multiple antibodies.Each antibody that specifically binds to a different epitope has adifferent structure. Thus, one antigen may have more than onecorresponding antibody. An antibody includes a full-lengthimmunoglobulin molecule or an immunologically active portion of afull-length immunoglobulin molecule, i.e., a molecule that contains anantigen binding site that immunospecifically binds an antigen of atarget of interest or part thereof, such targets including but notlimited to, cancer cell or cells that produce autoimmune antibodiesassociated with an autoimmune disease. The immunoglobulin disclosedherein can be of any type (e.g., IgG, IgE, IgM, IgD, and IgA), class(e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) or subclass ofimmunoglobulin molecule. The immunoglobulins can be derived from anyspecies such as human, murine, or rabbit. For the structure andproperties of the different classes of antibodies, see, e.g., Basic andClinical Immunology, 8th edition, Daniel P. Stites, Abba I. Terr andTristram G. Parslow (eds.), Appleton & Lange, Norwalk, Conn., 1994, page71 and Chapter 6.

“Antibody fragments” comprise a portion of a full length antibody,generally the antigen binding or variable region thereof. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂, and Fv fragments;diabodies; linear antibodies; minibodies (U.S. Pat. No. 5,641,870,Example 2; Zapata et al (1995) Protein Eng. 8(10): 1057-1062); Olafsenet al (2004) Protein Eng. Design & Sel. 17(4):315-323), fragmentsproduced by a Fab expression library, anti-idiotypic (anti-Id)antibodies, CDR (complementary determining region), and epitope-bindingfragments of any of the above which immunospecifically bind to cancercell antigens, viral antigens or microbial antigens, single-chainantibody molecules; and multispecific antibodies formed from antibodyfragments.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast to polyclonalantibody preparations which include different antibodies directedagainst different determinants (epitopes), each monoclonal antibody isdirected against a single determinant on the antigen. In addition totheir specificity, the monoclonal antibodies are advantageous in thatthey may be synthesized uncontaminated by other antibodies. The modifier“monoclonal” indicates the character of the antibody as being obtainedfrom a substantially homogeneous population of antibodies, and is not tobe construed as requiring production of the antibody by any particularmethod. For example, the monoclonal antibodies to be used in accordancewith the present invention may be made by the hybridoma method firstdescribed by Kohler et al (1975) Nature 256:495, or may be made byrecombinant DNA methods (see for example: U.S. Pat. No. 4,816,567; U.S.Pat. No. 5,807,715). In the hybridoma method, a mouse or otherappropriate host animal, such as a hamster, is immunized as describedabove to elicit lymphocytes that produce or are capable of producingantibodies that will specifically bind to the protein used forimmunization. Alternatively, lymphocytes may be immunized in vitro.After immunization, lymphocytes are isolated and then fused with amyeloma cell line using a suitable fusing agent, such as polyethyleneglycol, to form a hybridoma cell (Goding, (1986) Monoclonal Antibodies:Principles and Practice, pp. 59-103 Academic Press). The monoclonalantibodies may also be isolated from phage antibody libraries using thetechniques described in Clackson et al (1991) Nature, 352:624-628; Markset al (1991) J. Mol. Biol., 222:581-597.

The DNA that encodes the antibody may be modified to produce chimeric orfusion antibody polypeptides, for example, by substituting human heavychain and light chain constant domain (C_(H) and C_(L)) sequences forthe homologous murine sequences (U.S. Pat. No. 4,816,567; and Morrison,et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by fusing theimmunoglobulin coding sequence with all or part of the coding sequencefor a non-immunoglobulin polypeptide (heterologous polypeptide). Thenon-immunoglobulin polypeptide sequences can substitute for the constantdomains of an antibody, or they are substituted for the variable domainsof one antigen-combining site of an antibody to create a chimericbivalent antibody comprising one antigen-combining site havingspecificity for an antigen and another antigen-combining site havingspecificity for a different antigen.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend. The constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light-chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al(1984) Proc. Natl. Acad. Sci. USA, 81:6851-6855). Chimeric antibodies ofinterest herein include “primatized” antibodies comprising variabledomain antigen-binding sequences derived from a non-human primate (e.g.,Old World Monkey, Ape etc) and human constant region sequences.

“Humanized” forms of non-human (e.g., rodent) antibodies are chimericantibodies that contain minimal sequence derived from the non-humanantibody. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which residues from ahypervariable region of the recipient are replaced by residues from ahypervariable region of a non-human species (donor antibody) such asmouse, rat, rabbit or non-human primate having the desired antibodyspecificity, affinity, and capability. In some instances, frameworkregion (FR) residues of the human immunoglobulin are replaced bycorresponding non-human residues. Furthermore, humanized antibodies maycomprise residues that are not found in the recipient antibody or in thedonor antibody. These modifications are made to further refine antibodyperformance. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin and all, or substantially all, ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. The Fc fragment comprises the carboxy-terminal portionsof both H chains held together by disulfides. The effector functions ofantibodies are determined by sequences in the Fc region, which region isalso the part recognized by Fc receptors (FcR) found on certain types ofcells (Jones et al (1986) Nature 321:522-525; Riechmann et al (1988)Nature 332:323-329; Presta, (1992) Curr. Op. Struct. Biol. 2:593-596;Verhoeyen et al (1988) Science, 239:1534-1536; Sims et al (1993) J.Immunol. 151:2296; Chothia et al (1987) J. Mol. Biol., 196:901). Othermethods use a particular framework region derived from the consensussequence of all human antibodies of a particular subgroup of light orheavy chains (Carter et al (1992) Proc. Natl. Acad. Sci. USA, 89:4285;Presta et al (1993) J. Immunol. 151:2623).

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Transgenic animals (e.g., mice) are available that arecapable, upon immunization, of producing a full repertoire of humanantibodies in the absence of endogenous immunoglobulin production. Forexample, it has been described that the homozygous deletion of theantibody heavy-chain joining region (J_(H)) gene in chimeric andgerm-line mutant mice results in complete inhibition of endogenousantibody production. Transfer of the human germ-line immunoglobulin genearray into such germ-line mutant mice will result in the production ofhuman antibodies upon antigen challenge (Jakobovits et al (1993) Proc.Natl. Acad. Sci. USA, 90:2551; Jakobovits et al (1993) Nature,362:255-258; Bruggemann et al (1993) Year in Immuno. 7:33; U.S. Pat. No.5,545,806; U.S. Pat. No. 5,569,825; U.S. Pat. No. 5,591,669; U.S. Pat.No. 5,545,807; and WO 97/17852.

An “affinity matured” antibody is one with one or more alterations inone or more CDRs thereof which result in an improvement in the affinityof the antibody for antigen, compared to an antibody which does notpossess those alteration(s). Preferred affinity matured antibodies willhave nanomolar or even picomolar affinities for the target antigen.Affinity matured antibodies are produced affinity maturation by VH andVL domain shuffling (Marks et al (1992) Bio/Technology 10:779-783), orrandom mutagenesis of CDR and/or framework residues (Barbas et al (1994)Proc Nat. Acad. Sci, USA 91:3809-3813; Schier et al (1995) Gene169:147-155; Yelton et al (1995) J. Immunol. 155:1994-2004; Jackson etal (1995) J. Immunol. 154(7):3310-9; and Hawkins et al (1992) J. Mol.Biol. 226:889-896).

An “intact antibody” herein is one comprising VL and VH domains, as wellas a light chain constant domain (CL) and heavy chain constant domains,CH1, CH2 and CH3. The constant domains may be native sequence constantdomains (e.g., human native sequence constant domains) or amino acidsequence variant thereof. The intact antibody may have one or more“effector functions” which refer to those biological activitiesattributable to the Fc constant region (a native sequence Fc region oramino acid sequence variant Fc region) of an antibody. Examples ofantibody effector functions include Clq binding; complement dependentcytotoxicity; Fc receptor binding; antibody-dependent cell-mediatedcytotoxicity (ADCC); phagocytosis; and down regulation of cell surfacereceptors such as B cell receptor and BCR.

The term “amino acid sequence variant” refers to polypeptides havingamino acid sequences that differ to some extent from a native sequencepolypeptide. Ordinarily, amino acid sequence variants will possess atleast about 70% sequence identity with at least one receptor bindingdomain of a native sequence polypeptide or with at least one ligandbinding domain of a native receptor, and preferably, they will be atleast about 80%, more preferably, at least about 90% homologous bysequence with such receptor or ligand binding domains. The amino acidsequence variants possess substitutions, deletions, and/or insertions atcertain positions within the amino acid sequence of the native aminoacid sequence. Amino acids are designated by the conventional names,one-letter and three-letter codes.

“Sequence identity” is defined as the percentage of residues in theamino acid sequence variant that are identical after aligning thesequences and introducing gaps, if necessary, to achieve the maximumpercent sequence identity. Methods and computer programs for thealignment are well known in the art. One such computer program is “Align2,” authored by Genentech, Inc., which was filed with user documentationin the United States Copyright Office, Washington, D.C. 20559, on Dec.10, 1991, and which code is found in PCT/US03/07209 (U.S. Pat. No.7,288,248).

“Antibody-dependent cell-mediated cytotoxicity” and “ADCC” refer to acell-mediated reaction in which nonspecific cytotoxic cells that expressFc receptors (FcRs) (e.g., Natural Killer (NK) cells, neutrophils, andmacrophages) recognize bound antibody on a target cell and subsequentlycause lysis of the target cell. The primary cells for mediating ADCC, NKcells, express FcγRIII only, whereas monocytes express FcγRI, FcγRII andFcγRIII. FcR expression on hematopoietic cells in summarized is Table 3on page 464 of Ravetch and Kinet, (1991) “Annu. Rev. Immunol.” 9:457-92.To assess ADCC activity of a molecule of interest, an in vitro ADCCassay, such as that described in U.S. Pat. No. 5,500,362 and U.S. Pat.No. 5,821,337 may be performed. Useful effector cells for such assaysinclude peripheral blood mononuclear cells (PBMC) and Natural Killer(NK) cells. Alternatively, or additionally, ADCC activity of themolecule of interest may be assessed in vivo, e.g., in an animal modelsuch as that disclosed in Clynes et al (1998) Proc. Nat. Acad. Sci.(USA) 95:652-656.

“Human effector cells” are leukocytes which express one or more constantregion receptors (FcRs) and perform effector functions. Preferably, thecells express at least FcγRIII and perform ADCC effector function.Examples of human leukocytes which mediate ADCC include peripheral bloodmononuclear cells (PBMC), natural killer (NK) cells, monocytes,cytotoxic T cells and neutrophils; with PBMCs and NK cells beingpreferred. The effector cells may be isolated from a native sourcethereof, e.g., from blood or PBMCs as described herein.

The terms “Fc receptor” or “FcR” mean a receptor that binds to the Fcconstant region of an antibody. The preferred FcR is a native sequencehuman FcR. Moreover, a preferred FcR is one which binds an IgG antibody(a gamma receptor) and includes receptors of the FcγRI, FcγRII, andFcγRIII subclasses, including allelic variants and alternatively splicedforms of these receptors. FcγRII receptors include FcγRIIA (an“activating receptor”) and FcγRIIB (an “inhibiting receptor”), whichhave similar amino acid sequences that differ primarily in thecytoplasmic domains thereof. Activating receptor FcγRIIA contains animmunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmicdomain. Inhibiting receptor FcγRIIB contains an immunoreceptortyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (Seereview M. in Daëron, (1997) “Annu. Rev. Immunol.” 15:203-234). FcRs arereviewed in Ravetch and Kinet, (1991) “Annu. Rev. Immunol”., 9:457-92;Capel et al (1994) Immunomethods 4:25-34; and de Haas et al (1995) J.Lab. Clin. Med. 126:330-41. Other FcRs, including those to be identifiedin the future, are encompassed by the term “FcR” herein. The term alsoincludes the neonatal receptor, FcRn, which is responsible for thetransfer of maternal IgGs to the fetus (Guyer et al (1976) J. Immunol.,117:587 and Kim et al (1994) J. Immunol. 24:249).

“Complement dependent cytotoxicity” or “CDC” refers to the lysis of atarget cell in the presence of complement. Activation of the classicalcomplement pathway is initiated by the binding of the first component ofthe complement system (C1q) to antibodies (of the appropriate subclass)which are bound to their cognate antigen (Gazzano-Santoro et al (1996)J. Immunol. Methods 202:163).

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the framework regions (FRs). The variabledomains of native heavy and light chains each comprise four FRs, largelyadopting a β-sheet configuration, connected by three hypervariableregions, which form loops connecting, and in some cases forming part of,the β-sheet structure. The hypervariable regions in each chain are heldtogether in close proximity by the FRs and, with the hypervariableregions from the other chain, contribute to the formation of theantigen-binding site of antibodies (see Kabat et al (1991) Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md.). The constant domains arenot involved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody dependent cellular cytotoxicity (ADCC).

The term “hypervariable region”, “HVR”, or “HV”, when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six hypervariable regions; three in the VH (H1, H2, H3), andthree in the VL (L1, L2, L3). A number of hypervariable regiondelineations are in use and are encompassed herein. The KabatComplementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The “contact” hypervariable regions arebased on an analysis of the available complex crystal structures. Theresidues from each of these hypervariable regions are noted below.Unless otherwise denoted, Kabat numbering according to the KabatDatabase of aligned sequences of proteins will be employed (Wu and Kabat(1970) J. Exp. Med. 132:211-250; Johnson and Wu (2000) Nuc. Acids Res.28(1):214-218). Hypervariable region locations are generally as follows:amino acids 24-34 (HVR-L), amino acids 49-56 (HVR-L2), amino acids 89-97(HVR-L3), amino acids 26-35A (HVR-H1), amino acids 49-65 (HVR-H2), andamino acids 93-102 (HVR-H3). Hypervariable regions may also comprise“extended hypervariable regions” as follows: amino acids 24-36 (L1), andamino acids 46-56 (L2) in the VL. The variable domain residues arenumbered according to Kabat et al., supra for each of these definitions.An “altered hypervariable region” for the purposes herein is ahypervariable region comprising one or more (e.g. one to about 16) aminoacid substitution(s) therein. An “un-modified hypervariable region” forthe purposes herein is a hypervariable region having the same amino acidsequence as a non-human antibody from which it was derived, i.e. onewhich lacks one or more amino acid substitutions therein.

The terms “variable domain residue numbering as in Kabat”, “amino acidposition numbering as in Kabat”, and variations thereof, refer to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md. (1991). Using thisnumbering system, the actual linear amino acid sequence may containfewer or additional amino acids corresponding to a shortening of, orinsertion into, an FR or CDR of the variable domain. For example, aheavy chain variable domain may include a single amino acid insert(residue 52a according to Kabat) after residue 52 of H2 and insertedresidues (e.g. residues 82a, 82b, and 82c, etc according to Kabat) afterheavy chain FR residue 82. The Kabat numbering of residues may bedetermined for a given antibody by alignment at regions of homology ofthe sequence of the antibody with a “standard” Kabat numbered sequence.

“Binding affinity” generally refers to the strength of the sum total ofnoncovalent interactions between a single binding site of a molecule(e.g., an antibody) and its binding partner (e.g., an antigen). Unlessindicated otherwise, as used herein, “binding affinity” refers tointrinsic binding affinity which reflects a 1:1 interaction betweenmembers of a binding pair (e.g., antibody and antigen). The affinity ofa molecule X for its partner Y can generally be represented by thedissociation constant (Kd). Affinity can be measured by common methodsknown in the art, including those described herein. Low-affinityantibodies generally bind antigen slowly and tend to dissociate readily,whereas high-affinity antibodies generally bind antigen faster and tendto remain bound longer. A variety of methods of measuring bindingaffinity are known in the art, any of which can be used for purposes ofthe present invention. Specific illustrative embodiments are describedin the following.

An “antigen” is a predetermined polypeptide, carbohydrate, nucleic acid,lipid, hapten or other naturally occurring or synthetic compound towhich an antibody can selectively bind.

“Framework” or “FR” residues are those variable domain residues otherthan the hypervariable region residues as herein defined. A “humanconsensus framework” is a framework which represents the most commonlyoccurring amino acid residue in a selection of human immunoglobulin VLor VH framework sequences. Generally, the selection of humanimmunoglobulin VL or VH sequences is from a subgroup of variable domainsequences. Generally, the subgroup of sequences is a subgroup as inKabat et al, Sequences of Proteins of Immunological Interest, 5th Ed.Public Health Service, National Institutes of Health, Bethesda, Md.(1991). In one embodiment, for the VL, the subgroup is subgroup kappa Ias in Kabat et al. In one embodiment, for the VH, the subgroup issubgroup III as in Kabat et al. A “VH subgroup III consensus framework”comprises the consensus sequence obtained from the amino acid sequencesin variable heavy subgroup III of Kabat et al. A “VL subgroup Iconsensus framework” comprises the consensus sequence obtained from theamino acid sequences in variable light kappa subgroup I of Kabat et al.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (CH1) of the heavy chain. Fab′ fragmentsdiffer from Fab fragments by the addition of a few residues at thecarboxy terminus of the heavy chain CH1 domain including one or morecysteines from the antibody hinge region. Fab′-SH is the designationherein for Fab′ in which the cysteine residue(s) of the constant domainsbear at least one free thiol group. F(ab′)2 antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

The “light chains” of antibodies from any vertebrate species can beassigned to one of two clearly distinct types, called kappa (K) andlambda (X), based on the amino acid sequences of their constant domains.

“Single-chain Fv” or “scFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain. Preferably, the Fv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding (Plückthun in ThePharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Mooreeds., Springer-Verlag, New York, pp. 269-315 (1994).

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a variable heavy domain(VH) connected to a variable light domain (VL) in the same polypeptidechain (VH-VL). By using a linker that is too short to allow pairingbetween the two domains on the same chain, the domains are forced topair with the complementary domains of another chain and create twoantigen-binding sites (EP 404,097; WO 93/11161; Hollinger et al (1993)Proc. Natl. Acad. Sci. USA 90:6444-6448).

A “free cysteine amino acid” refers to a cysteine amino acid residuewhich has been engineered into a parent antibody, has a thiol functionalgroup (—SH), and is not paired as, or otherwise part of, anintramolecular or intermolecular disulfide bridge.

The term “thiol reactivity value” is a quantitative characterization ofthe reactivity of free cysteine amino acids. The thiol reactivity valueis the percentage of a free cysteine amino acid in a cysteine engineeredantibody which reacts with a thiol-reactive reagent, and converted to amaximum value of 1. For example, a free cysteine amino acid on acysteine engineered antibody which reacts in 100% yield with athiol-reactive reagent, such as a biotin-maleimide reagent, to form abiotin-labelled antibody has a thiol reactivity value of 1.0. Anothercysteine amino acid engineered into the same or different parentantibody which reacts in 80% yield with a thiol-reactive reagent has athiol reactivity value of 0.8. Another cysteine amino acid engineeredinto the same or different parent antibody which fails totally to reactwith a thiol-reactive reagent has a thiol reactivity value of 0.Determination of the thiol reactivity value of a particular cysteine maybe conducted by ELISA assay, mass spectroscopy, liquid chromatography,autoradiography, or other quantitative analytical tests. Thiol-reactivereagents which allow capture of the cysteine engineered antibody andcomparison and quantitation of the cysteine reactivity includebiotin-PEO-maleimide((+)-biotinyl-3-maleimidopropionamidyl-3,6-dioxaoctainediamine, Oda etal (2001) Nature Biotechnology 19:379-382, Pierce Biotechnology, Inc.)Biotin-BMCC, PEO-Iodoacetyl Biotin, Iodoacetyl-LC-Biotin, andBiotin-HPDP (Pierce Biotechnology, Inc.), andNao-(3-maleimidylpropionyl)biocytin (MPB, Molecular Probes, Eugene,Oreg.). Other commercial sources for biotinylation, bifunctional andmultifunctional linker reagents include Molecular Probes, Eugene, Oreg.,and Sigma, St. Louis, Mo.

A “parent antibody” is an antibody comprising an amino acid sequencefrom which one or more amino acid residues are replaced by one or morecysteine residues. The parent antibody may comprise a native or wildtype sequence. The parent antibody may have pre-existing amino acidsequence modifications (such as additions, deletions and/orsubstitutions) relative to other native, wild type, or modified forms ofan antibody. A parent antibody may be directed against a target antigenof interest, e.g. a biologically important polypeptide. Antibodiesdirected against nonpolypeptide antigens (such as tumor-associatedglycolipid antigens; see U.S. Pat. No. 5,091,178) are also contemplated.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

An antibody “which binds” a molecular target or an antigen of interest,e.g., TENB2 or CA125 antigens, is one capable of binding that antigenwith sufficient affinity such that the antibody is useful in targeting acell expressing the antigen. Where the antibody is one which bindsTENB2, it will usually preferentially bind TENB2, and may be one whichdoes not significantly cross-react with other proteins. In suchembodiments, the extent of binding of the antibody to these non-TENB2proteins (e.g., cell surface binding to endogenous receptor) will beless than 10% as determined by fluorescence activated cell sorting(FACS) analysis or radioimmunoprecipitation (RIA).

“Treating” or “treatment” or “alleviation” refers to both therapeutictreatment and prophylactic or preventative measures, wherein the objectis to prevent or slow down (lessen) the targeted pathologic condition ordisorder. Those in need of treatment include those already with thedisorder as well as those prone to have the disorder or those in whomthe disorder is to be prevented. A subject or mammal is successfully“treated” for a CA125/O0772P polypeptide-expressing cancer if, afterreceiving a therapeutic amount of an anti-CA125/O772P antibody, such asa cysteine engineered anti-TENB2 antibody, or antibody drug conjugatethereof, according to the methods of the present invention, the patientshows observable and/or measurable reduction in or absence of one ormore of the following: reduction in the number of cancer cells orabsence of the cancer cells; reduction in the tumor size; inhibition(i.e., slow to some extent and preferably stop) of cancer cellinfiltration into peripheral organs including the spread of cancer intosoft tissue and bone; inhibition (i.e., slow to some extent andpreferably stop) of tumor metastasis; inhibition, to some extent, oftumor growth; and/or relief to some extent, one or more of the symptomsassociated with the specific cancer; reduced morbidity and mortality,and improvement in quality of life issues. To the extent the cysteineengineered anti-TENB2 antibody, or antibody drug conjugate thereof, mayprevent growth and/or kill existing cancer cells, it may be cytostaticand/or cytotoxic. Reduction of these signs or symptoms may also be feltby the patient. The above parameters for assessing successful treatmentand improvement in the disease are readily measurable by routineprocedures familiar to a physician. For cancer therapy, efficacy can bemeasured, for example, by assessing the time to disease progression(TTP) and/or determining the response rate (RR). Metastasis can bedetermined by staging tests and by bone scan and tests for calcium leveland other enzymes to determine spread to the bone. CT scans can also bedone to look for spread to the pelvis and lymph nodes in the area. ChestX-rays and measurement of liver enzyme levels by known methods are usedto look for metastasis to the lungs and liver, respectively. Otherroutine methods for monitoring the disease include transrectalultrasonography (TRUS) and transrectal needle biopsy (TRNB).

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. A “tumor” comprises one or more cancerouscells, and refers to all neoplastic cell growth and proliferation,whether malignant or benign, and all pre-cancerous and cancerous cellsand tissues. Examples of cancer include, but are not limited to,carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include squamouscell cancer (e.g., epithelial squamous cell cancer), lung cancerincluding small-cell lung cancer, non-small cell lung cancer (“NSCLC”),adenocarcinoma of the lung and squamous carcinoma of the lung, cancer ofthe peritoneum, hepatocellular cancer, gastric or stomach cancerincluding gastrointestinal cancer, pancreatic cancer, glioblastoma,cervical cancer, ovarian cancer, liver cancer, bladder cancer, hepatoma,breast cancer, colon cancer, rectal cancer, colorectal cancer,endometrial or uterine carcinoma, salivary gland carcinoma, kidney orrenal cancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, anal carcinoma, penile carcinoma, as well as head and neckcancer.

A cancer which “overexpresses” an antigenic receptor is one which hassignificantly higher levels of the receptor, such as TENB2, at the cellsurface thereof, compared to a noncancerous cell of the same tissuetype. Such overexpression may be caused by gene amplification or byincreased transcription or translation. Receptor overexpression may bedetermined in a diagnostic or prognostic assay by evaluating increasedlevels of the receptor protein present on the surface of a cell (e.g.,via an immunohistochemistry assay; IHC). Alternatively, or additionally,one may measure levels of receptor-encoding nucleic acid in the cell,e.g., via fluorescent in situ hybridization (FISH; see WO 98/45479),southern blotting, or polymerase chain reaction (PCR) techniques, suchas real time quantitative reverse-transcriptase PCR (qRT-PCR).

“Human effector cells” are leukocytes which express one or more FcRs andperform effector functions. Preferably, the cells express at leastFcγRIII and perform ADCC effector function. Examples of human leukocyteswhich mediate ADCC include peripheral blood mononuclear cells (PBMC),natural killer (NK) cells, monocytes, cytotoxic T cells and neutrophils;with PBMCs and NK cells being preferred. The effector cells may beisolated from a native source, e.g., from blood.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer.

The term “therapeutically effective amount” refers to an amount of adrug, e.g. a cysteine engineered anti-TENB2 antibody drug conjugate orchemotherapeutic agent, effective to treat a disease or disorder in amammal. In the case of cancer, the therapeutically effective amount ofthe drug may reduce the number of cancer cells; reduce the tumor size;inhibit (i.e., slow to some extent and preferably stop) cancer cellinfiltration into peripheral organs; inhibit (i.e., slow to some extentand preferably stop) tumor metastasis; inhibit, to some extent, tumorgrowth; and/or relieve to some extent one or more of the symptomsassociated with the cancer. To the extent the drug may prevent growthand/or kill existing cancer cells, it may be cytostatic and/orcytotoxic. The term “cytostatic” refers to the effect of limiting thefunction of cells, such as limiting cellular growth or proliferation ofcells. For cancer therapy, efficacy can, for example, be measured byassessing the time to disease progression (TTP) and/or determining theresponse rate (RR).

A “chemotherapeutic agent” is a chemical compound useful in thetreatment of cancer. Examples of chemotherapeutic agents includeerlotinib (TARCEVA®, Genentech/OSI Pharm.), bortezomib (VELCADE®,Millenium Pharm.), fulvestrant (FASLODEX@, AstraZeneca), sutent (SU11248, Pfizer), letrozole (FEMARA®, Novartis), imatinib mesylate(GLEEVEC®, Novartis), PTK787/ZK 222584 (Novartis), oxaliplatin(Eloxatin®, Sanofi), 5-FU (5-fluorouracil), leucovorin, Rapamycin(Sirolimus, RAPAMUNE®, Wyeth), lapatinib (TYKERB®, GSK572016,GlaxoSmithKline), lonafarnib (SCH 66336), sorafenib (BAY43-9006, BayerLabs.), and gefitinib (IRESSA@, Astrazeneca), AG1478, AG1571 (SU 5271;Sugen), alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (including thesynthetic analogue topotecan); bryostatin; callystatin; CC-1065(including its adozelesin, carzelesin and bizelesin syntheticanalogues); cryptophycins (particularly cryptophycin 1 and cryptophycin8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189and CB 1-TM1); eleutherobin; pancratistatin; a sarcodictyin;spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine,cholophosphamide, estramustine, ifosfamide, mechlorethamine,mechlorethamine oxide hydrochloride, melphalan, novembichin,phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureassuch as carmustine, chlorozotocin, fotemustine, lomustine, nimustine,and ranimnustine; antibiotics such as the enediyne antibiotics (e. g.,calicheamicin, especially calicheamicin gamma1I and calicheamicinomegaI1 (Angew Chem Intl. Ed. Engl. (1994) 33:183-186); dynemicin,including dynemicin A; bisphosphonates, such as clodronate; anesperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, anthramycin, azaserine, bleomycins, cactinomycin,carabicin, carminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®doxorubicin (including morpholino-doxorubicin,cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin anddeoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin,mitomycins such as mitomycin C, mycophenolic acid, nogalamycin,olivomycins, peplomycin, potfiromycin, puromycin, quelamycin,rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex,zinostatin, zorubicin; anti-metabolites such as methotrexate and5-fluorouracil (5-FU); folic acid analogues such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofiran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g.,paclitaxel (TAXOL®, Bristol-Myers Squibb Oncology, Princeton, N.J.),ABRAXANE™Cremophor-free, albumin-engineered nanoparticle formulation ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® doxetaxel (Rhône-Poulenc Rorer, Antony, France); chloranbucil;GEMZAR® gemcitabine; 6-thioguanine; mercaptopurine; methotrexate;platinum analogs such as cisplatin and carboplatin; vinblastine;platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine;NAVELBINE® vinorelbine; novantrone; teniposide; edatrexate; daunomycin;aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluorometlhylornithine (DMFO); retinoids such as retinoic acid;capecitabine; and pharmaceutically acceptable salts, acids orderivatives of any of the above.

Also included in this definition of “chemotherapeutic agent” are: (i)anti-hormonal agents that act to regulate or inhibit hormone action ontumors such as anti-estrogens and selective estrogen receptor modulators(SERMs), including, for example, tamoxifen (including NOLVADEX®tamoxifen), raloxifene, droloxifene, 4-hydroxytamoxifen, trioxifene,keoxifene, LY 117018, onapristone, and FARESTON. toremifene; (ii)aromatase inhibitors that inhibit the enzyme aromatase, which regulatesestrogen production in the adrenal glands, such as, for example,4(5)-imidazoles, aminoglutethimide, MEGASE® megestrol acetate, AROMASIN®exemestane, formestanie, fadrozole, RIVISOR® vorozole, FEMARA®letrozole, and ARIMIDEX® anastrozole; (iii) anti-androgens such asflutamide, nilutamide, bicalutamide, leuprolide, and goserelin; as wellas troxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv)aromatase inhibitors; (v) protein kinase inhibitors; (vi) lipid kinaseinhibitors; (vii) antisense oligonucleotides, particularly those whichinhibit expression of genes in signaling pathways implicated in abherantcell proliferation, such as, for example, PKC-alpha, Ralf and H-Ras;(viii) ribozymes such as a VEGF expression inhibitor (e.g., ANGIOZYME®ribozyme) and a HER2 expression inhibitor; (ix) vaccines such as genetherapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine,and VAXID® vaccine; PROLEUKIN® rIL-2; LURTOTECAN® topoisomerase 1inhibitor; ABARELIX® rmRH; (x) anti-angiogenic agents such asbevacizumab (AVASTIN®, Genentech); and (xi) pharmaceutically acceptablesalts, acids or derivatives of any of the above.

The term “cytokine” is a generic term for proteins released by one cellpopulation which act on another cell as intercellular mediators.Examples of such cytokines are lymphokines, monokines, and traditionalpolypeptide hormones. Included among the cytokines are growth hormonesuch as human growth hormone, N-methionyl human growth hormone, andbovine growth hormone; parathyroid hormone; thyroxine; insulin;proinsulin; relaxin; prorelaxin; glycoprotein hormones such as folliclestimulating hormone (FSH), thyroid stimulating hormone (TSH), andluteinizing hormone (LH); hepatic growth factor; fibroblast growthfactor; prolactin; placental lactogen; tumor necrosis factor-α and -β;mullerian-inhibiting substance; mouse gonadotropin-associated peptide;inhibin; activin; vascular endothelial growth factor; integrin;thrombopoietin (TPO); nerve growth factors such as NGF-β;platelet-growth factor; transforming growth factors (TGFs) such as TGF-αand TGF-β; insulin-like growth factor-I and -II; erythropoietin (EPO);osteoinductive factors; interferons such as interferon-α, -β, and -γ;colony stimulating factors (CSFs) such as macrophage-CSF (M-CSF);granulocyte-macrophage-CSF (GM-CSF); and granulocyte-CSF (G-CSF);interleukins (ILs) such as IL-1, IL-1α, IL-2, IL-3, IL-4, IL-5, IL-6,IL-7, IL-8, IL-9, IL-10, IL-11, IL-12; a tumor necrosis factor such asTNF-α or TNF-β; and other polypeptide factors including LIF and kitligand (KL). As used herein, the term cytokine includes proteins fromnatural sources or from recombinant cell culture and biologically activeequivalents of the native sequence cytokines.

The term “label” means any moiety which can be covalently attached to anantibody and that functions to: (i) provide a detectable signal; (ii)interact with a second label to modify the detectable signal provided bythe first or second label, e.g. FRET (fluorescence resonance energytransfer); (iii) stabilize interactions or increase affinity of binding,with antigen or ligand; (iv) affect mobility, e.g. electrophoreticmobility, or cell-permeability, by charge, hydrophobicity, shape, orother physical parameters, or (v) provide a capture moiety, to modulateligand affinity, antibody/antigen binding, or ionic complexation.

The phrase “pharmaceutically acceptable salt,” as used herein, refers topharmaceutically acceptable organic or inorganic salts of an ADC.Exemplary salts include, but are not limited, to sulfate, citrate,acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate,phosphate, acid phosphate, isonicotinate, lactate, salicylate, acidcitrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate,succinate, maleate, gentisinate, fumarate, gluconate, glucuronate,saccharate, formate, benzoate, glutamate, methanesulfonate,ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and pamoate(i.e., 1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Apharmaceutically acceptable salt may involve the inclusion of anothermolecule such as an acetate ion, a succinate ion or other counterion.The counterion may be any organic or inorganic moiety that stabilizesthe charge on the compound. Furthermore, a pharmaceutically acceptablesalt may have more than one charged atom in its structure. Instanceswhere multiple charged atoms are part of the pharmaceutically acceptablesalt can have multiple counter ions. Hence, a pharmaceuticallyacceptable salt can have one or more charged atoms and/or one or morecounterion.

“Pharmaceutically acceptable solvate” refers to an association of one ormore solvent molecules and an ADC. Examples of solvents that formpharmaceutically acceptable solvates include, but are not limited to,water, isopropanol, ethanol, methanol, DMSO, ethyl acetate, acetic acid,and ethanolamine.

“Carriers” as used herein include pharmaceutically acceptable carriers,excipients, or stabilizers which are nontoxic to the cell or mammalbeing exposed thereto at the dosages and concentrations employed. Oftenthe physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN®, polyethylene glycol (PEG), and PLURONICS®.

Stereochemical definitions and conventions used herein generally followS. P. Parker, Ed., McGraw-Hill Dictionary of Chemical Terms (1984)McGraw-Hill Book Company, New York; and Eliel, E. and Wilen, S.,Stereochemistry of Organic Compounds (1994) John Wiley & Sons, Inc., NewYork. Many organic compounds exist in optically active forms, i.e., theyhave the ability to rotate the plane of plane-polarized light. Indescribing an optically active compound, the prefixes D and L, or R andS, are used to denote the absolute configuration of the molecule aboutits chiral center(s). The prefixes d and l or (+) and (−) are employedto designate the sign of rotation of plane-polarized light by thecompound, with (−) or 1 meaning that the compound is levorotatory. Acompound prefixed with (+) or d is dextrorotatory. For a given chemicalstructure, these stereoisomers are identical except that they are mirrorimages of one another. A specific stereoisomer may also be referred toas an enantiomer, and a mixture of such isomers is often called anenantiomeric mixture. A 50:50 mixture of enantiomers is referred to as aracemic mixture or a racemate, which may occur where there has been nostereoselection or stereospecificity in a chemical reaction or process.The terms “racemic mixture” and “racemate” refer to an equimolar mixtureof two enantiomeric species, devoid of optical activity.

The following abbreviations are used herein and have the indicateddefinitions: BME is beta-mercaptoethanol, Boc is N-(t-butoxycarbonyl),cit is citrulline (2-amino-5-ureido pentanoic acid), dap is dolaproine,DCC is 1,3-dicyclohexylcarbodiimide, DCM is dichloromethane, DEA isdiethylamine, DEAD is diethylazodicarboxylate, DEPC isdiethylphosphorylcyanidate, DIAD is diisopropylazodicarboxylate, DIEA isN,N-diisopropylethylamine, dil is dolaisoleucine, DMA isdimethylacetamide, DMAP is 4-dimethylaminopyridine, DME isethyleneglycol dimethyl ether (or 1,2-dimethoxyethane), DMF isN,N-dimethylformamide, DMSO is dimethylsulfoxide, doe is dolaphenine,dov is N,N-dimethylvaline, DTNB is 5,5′-dithiobis(2-nitrobenzoic acid),DTPA is diethylenetriaminepentaacetic acid, DTT is dithiothreitol, EDCIis 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, EEDQ is2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline, ES-MS is electrospraymass spectrometry, EtOAc is ethyl acetate, Fmoc isN-(9-fluorenylmethoxycarbonyl), gly is glycine, HATU isO-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate, HOBt is 1-hydroxybenzotriazole, HPLC is highpressure liquid chromatography, ile is isoleucine, lys is lysine, MeCN(CH₃CN) is acetonitrile, MeOH is methanol, Mtr is 4-anisyldiphenylmethyl(or 4-methoxytrityl), nor is (1S,2R)-(+)-norephedrine, PAB isp-aminobenzylcarbamoyl, PBS is phosphate-buffered saline (pH 7), PEG ispolyethylene glycol, Ph is phenyl, Pnp is p-nitrophenyl, MC is6-maleimidocaproyl, phe is L-phenylalanine, PyBrop is bromotris-pyrrolidino phosphonium hexafluorophosphate, SEC is size-exclusionchromatography, Su is succinimide, TFA is trifluoroacetic acid, TLC isthin layer chromatography, UV is ultraviolet, and val is valine.

Cysteine Engineered Anti-TENB2 Antibodies

The compounds of the invention include cysteine engineered anti-TENB2antibodies where one or more amino acids of any form of wild-type orparent anti-TENB2 antibody is replaced with a cysteine amino acid. Theengineered cysteine amino acid is a free cysteine acid and not part ofan intrachain or interchain disulfide unit. Any form of anti-TENB2antibody may be so engineered, i.e. mutated. For example, a parent Fabantibody fragment may be engineered to form a cysteine engineered Fab,referred to herein as “ThioFab.” Similarly, a parent monoclonal antibodymay be engineered to form a “ThioMab.” It should be noted that a singlesite mutation yields a single engineered cysteine residue in a ThioFab,while a single site mutation yields two engineered cysteine residues ina ThioMab, due to the dimeric nature of the IgG antibody. The cysteineengineered anti-TENB2 antibodies of the invention include monoclonalantibodies, humanized or chimeric monoclonal antibodies, antigen-bindingfragments of antibodies, fusion polypeptides and analogs thatpreferentially bind cell-associated TENB2 polypeptides.

Cysteine engineered anti-TENB2 antibodies retain the antigen bindingcapability of their wild type, parent anti-TENB2 antibody counterparts.Thus, cysteine engineered anti-TENB2 antibodies are capable of bindingto TENB2 antigens.

A cysteine engineered anti-TENB2 antibody comprises one or more freecysteine amino acids with reduced sulfhydryl (thiol) groups wherein thecysteine engineered anti-TENB2 antibody binds to a TENB2 polypeptide.

In one embodiment, the cysteine engineered anti-TENB2 antibody isprepared by a process comprising replacing one or more amino acidresidues of a parent anti-TENB2 antibody by cysteine.

Mutants with replaced (“engineered”) cysteine (Cys) residues may beevaluated for the reactivity of the newly introduced, engineeredcysteine thiol groups. The thiol reactivity value is a relative,numerical term in the range of 0 to 1.0 and can be measured for anycysteine engineered antibody. Thiol reactivity values of cysteineengineered antibodies of the invention may be in the ranges of 0.6 to1.0; 0.7 to 1.0; or 0.8 to 1.0.

In one aspect, the invention concerns an isolated cysteine engineeredanti-TENB2 antibody comprising an amino acid sequence that is encoded bya nucleotide sequence that hybridizes to the complement of a DNAmolecule encoding (a) a cysteine engineered antibody having afull-length amino acid sequence as disclosed herein, (b) a cysteineengineered antibody amino acid sequence lacking the signal peptide asdisclosed herein, (c) an extracellular domain of a transmembranecysteine engineered antibody protein, with or without the signalpeptide, as disclosed herein, (d) an amino acid sequence encoded by anyof the nucleic acid sequences disclosed herein or (e) any otherspecifically defined fragment of a full-length cysteine engineeredantibody amino acid sequence as disclosed herein.

In one aspect, the invention provides an isolated cysteine engineeredanti-TENB2 antibody without the N-terminal signal sequence and/orwithout the initiating methionine and is encoded by a nucleotidesequence that encodes such an amino acid sequence as described in.Processes for producing the same are also herein described, whereinthose processes comprise culturing a host cell comprising a vector whichcomprises the appropriate encoding nucleic acid molecule underconditions suitable for expression of the cysteine engineered antibodyand recovering the cysteine engineered antibody from the cell culture.

Another aspect of the invention provides an isolated cysteine engineeredanti-TENB2 antibody which is either transmembrane domain-deleted ortransmembrane domain-inactivated. Processes for producing the same arealso herein described, wherein those processes comprise culturing a hostcell comprising a vector which comprises the appropriate encodingnucleic acid molecule under conditions suitable for expression of thecysteine engineered antibody and recovering the cysteine engineeredantibody from the cell culture.

In other embodiments, the invention provides isolated anti-TENB2chimeric cysteine engineered antibodies comprising any of the hereindescribed cysteine engineered antibody fused to a heterologous(non-TENB2) polypeptide. Examples of such chimeric molecules compriseany of the herein described cysteine engineered antibodies fused to aheterologous polypeptide such as, for example, an epitope tag sequenceor an Fc region of an immunoglobulin.

The cysteine engineered anti-TENB2 antibody may be a monoclonalantibody, antibody fragment, chimeric antibody, humanized antibody,single-chain antibody or antibody that competitively inhibits thebinding of an anti-TENB2 polypeptide antibody to its respectiveantigenic epitope. Antibodies of the present invention may optionally beconjugated to a growth inhibitory agent or cytotoxic agent such as atoxin, including, for example, an auristatin, an antibiotic, aradioactive isotope, a nucleolytic enzyme, or the like.

The antibodies of the present invention may optionally be produced inCHO cells or bacterial cells and preferably inhibit the growth orproliferation of or induce the death of a cell to which they bind. Fordiagnostic purposes, the antibodies of the present invention may bedetectably labeled, attached to a solid support, or the like.

In other embodiments of the present invention, the invention providesvectors comprising DNA encoding any of the herein described cysteineengineered anti-TENB2 antibodies. Host cells comprising any such vectorare also provided. By way of example, the host cells may be CHO cells,E. coli cells, or yeast cells. A process for producing any of the hereindescribed polypeptides is further provided and comprises culturing hostcells under conditions suitable for expression of the desiredpolypeptide and recovering the desired polypeptide from the cellculture.

Parent and cysteine engineered anti-TENB2 antibodies bind to a TENB2polypeptide or TENB2 polypeptide variant described in PCT/US03/07209U.S. Pat. No. 7,288,248).

A TENB2 polypeptide variant is a TENB2 polypeptide having at least about80% amino acid sequence identity with a TENB2 which is a: (i)full-length native sequence; (ii) a polypeptide sequence lacking thesignal peptide; (iii) an extracellular domain, with or without thesignal peptide; (iv) or any other fragment of a full-length TENB2polypeptide sequence. Such TENB2 polypeptide variants include, forinstance, polypeptides wherein one or more amino acid residues areadded, or deleted, at the N- or C-terminus of the full-length nativeamino acid sequence. Ordinarily, a TENB2 polypeptide variant will haveat least about 80% amino acid sequence identity, alternatively at leastabout 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity, to afull-length native sequence TENB2 polypeptide sequence, a TENB2polypeptide sequence lacking the signal peptide, an extracellular domainof a TENB2 polypeptide, with or without the signal peptide, or any otherspecifically defined fragment of a full-length TENB2 polypeptidesequence. Ordinarily, TENB2 polypeptide variants are at least about 10amino acids in length, alternatively at least about 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210,220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350,360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490,500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600 amino acids inlength, or more. Optionally, TENB2 variant polypeptides will have nomore than one conservative amino acid substitution as compared to thenative TENB2 polypeptide sequence, alternatively no more than 2, 3, 4,5, 6, 7, 8, 9, or 10 conservative amino acid substitution as compared tothe native TENB2 polypeptide sequence.

TENB2 polypeptides may be prepared by recombinant expression in: (i) E.coli with pBR322 vector; (ii) mammalian cells such as human HEK293 cells(ATCC CCL 1573), COS (simian fibroblast, SV-40) cells, Chinese HamsterOvary (CHO) cells with the pRK5 vector; (iii) yeast, such as yeaststrain AB110; or (iv) baculovirus-infected insect cells (PCT/US03/07209;U.S. Pat. No. 7,288,248). Native or recombinant TENB2 polypeptides maybe purified by a variety of standard techniques in the art of proteinpurification. For example, pro-TENB2 polypeptide, mature TENB2polypeptide, or pre-TENB2 polypeptide is purified by immunoaffinitychromatography using antibodies specific for the TENB2 polypeptide ofinterest. In general, an immunoaffinity column is constructed bycovalently coupling the anti-TENB2 polypeptide antibody to an activatedchromatographic resin. TENB2 polypeptides may be produced recombinantlyas a fusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. Alternatively,TENB2 polypeptides may be produced as fusion polypeptides with a signalsequence and a heterologous polypeptide sequence that allowspurification of the TENB2 fusion polypeptide; examples of suchpolypeptides are polyhistidine (His₆ (SEQ ID NO: 24) or Hiss (SEQ ID NO:25)), human IgG Fc, the FLAG epitope (KDYKDDDDK (SEQ ID NO: 26)), andthe gD epitope (KYALADASLKMADPNRFRGKDLPVL (SEQ ID NO: 27)). The signalsequence may be a component of the vector, or it may be a part of theanti-TENB2 antibody- or TENB2 polypeptide-encoding DNA that is insertedinto the vector. The signal sequence may be a prokaryotic signalsequence selected, for example, from the group of the alkalinephosphatase, penicillinase, 1pp, or heat-stable enterotoxin II leaders.For yeast secretion the signal sequence may be, e.g., the yeastinvertase leader, alpha factor leader (including Saccharomyces andKluyveromyces α-factor leaders (U.S. Pat. No. 5,010,182), or acidphosphatase leader, the C. albicans glucoamylase leader (EP 0362179), orthe signal described in WO 90/13646. In mammalian cell expression,mammalian signal sequences may be used to direct secretion of theprotein, such as signal sequences from secreted polypeptides of the sameor related species, as well as viral secretory leaders.

A TENB2-expressing cell expresses an endogenous or transfected TENB2polypeptide antigen either on the cell surface or in a secreted form. ATENB2-expressing cancer comprises cells that have a TENB2 polypeptidepresent on the cell surface or that produce and secrete a TENB2antigenic polypeptide. A TENB2-expressing cancer optionally producessufficient levels of TENB2 polypeptide on the surface of cells thereof,such that an anti-TENB2 antibody, or antibody drug conjugate thereof,can bind thereto and may exert a therapeutic effect with respect to thecancer. A cancer which overexpresses a TENB2 polypeptide is one whichhas significantly higher levels of TENB2 polypeptide at the cell surfacethereof, or produces and secretes, compared to a noncancerous cell ofthe same tissue type. Such overexpression may be caused by geneamplification or by increased transcription or translation. TENB2polypeptide overexpression may be determined in a clinical setting byevaluating increased levels of the TENB2 protein present on the surfaceof a cell, or secreted by the cell (e.g., via an immunohistochemistryassay using anti-TENB2 antibodies prepared against an isolated TENB2polypeptide which may be prepared using recombinant DNA technology froman isolated nucleic acid encoding the TENB2 polypeptide; FACS analysis,etc.). Alternatively, or additionally, one may measure levels of TENB2polypeptide-encoding nucleic acid or mRNA in the cell, e.g., viafluorescent in situ hybridization (FISH) using a nucleic acid basedprobe corresponding to a TENB2-encoding nucleic acid or the complementthereof; (WO 98/45479), Southern blotting, Northern blotting, orpolymerase chain reaction (PCR) techniques, such as real timequantitative reverse-transcriptase PCR (qRT-PCR). One may also detectTENB2 polypeptide overexpression by measuring shed antigen in abiological fluid such as serum, e.g., using antibody-based assays (U.S.Pat. No. 4,933,294; WO 91/05264; U.S. Pat. No. 5,401,638; Sias et al(1990) J. Immunol. Methods 132:73-80). Various other in vivo assays maybe contemplated. Alternatively, cells within the body of the patient maybe exposed to an antibody which is optionally labeled with a detectablelabel, e.g., a radioactive isotope, and binding of the antibody to cellsin the patient can be evaluated, e.g., by external scanning forradioactivity or by analyzing a biopsy taken from a patient previouslyexposed to the antibody.

Parent and cysteine engineered anti-TENB2 antibodies are capable ofbinding, preferably specifically, to a TENB2 polypeptide as describedherein. TENB2 binding oligopeptides may be identified without undueexperimentation using well known techniques. In this regard, it is notedthat techniques for screening oligopeptide libraries for oligopeptidesthat are capable of specifically binding to a polypeptide target arewell known in the art (U.S. Pat. No. 5,556,762; U.S. Pat. No. 5,750,373;U.S. Pat. No. 4,708,871; U.S. Pat. No. 4,833,092; U.S. Pat. No.5,223,409; U.S. Pat. No. 5,403,484; U.S. Pat. No. 5,571,689; U.S. Pat.No. 5,663,143; WO 84/03506; WO84/03564; Geysen et al (1984) Proc. Natl.Acad. Sci. USA, 81:3998-4002; Geysen et al (1985) Proc. Natl. Acad. Sci.USA, 82:178-182; Geysen et al., in Synthetic Peptides as Antigens,130-149 (1986); Geysen et al., J. Immunol. Meth., 102:259-274 (1987);Schoofs et al., J. Immunol., 140:611-616 (1988), Cwirla, S. E. et al.(1990) Proc. Natl. Acad. Sci. USA, 87:6378; Lowman, H. B. et al. (1991)Biochemistry, 30:10832; Clackson, T. et al. (1991) Nature, 352: 624;Marks, J. D. et al. (1991), J. Mol. Biol., 222:581; Kang, A. S. et al.(1991) Proc. Natl. Acad. Sci. USA, 88:8363, and Smith, G. P. (1991)Current Opin. Biotechnol., 2:668).

The parent and cysteine engineered anti-TENB2 antibodies of theinvention include polyclonal, monoclonal, humanized, human, bispecific,and heteroconjugate antibodies. Various forms of a humanized anti-TENB2antibody are contemplated. For example, the humanized antibody may be anantibody fragment, such as a Fab. Alternatively, the humanized antibodymay be an intact antibody, such as an intact IgG1 antibody.

Bispecific anti-TENB2 antibodies are antibodies that have bindingspecificities for at least two different epitopes. Exemplary bispecificanti-TENB2 antibodies may bind to two different epitopes of a TENB2protein as described herein. Other such antibodies may combine a TENB2binding site with a binding site for another protein. Alternatively, ananti-TENB2 arm may be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g. CD3),or Fc receptors for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32) andFcγRIII (CD16), so as to focus and localize cellular defense mechanismsto the TENB2-expressing cell. Bispecific antibodies may also be used tolocalize cytotoxic agents to cells which express TENB2. These antibodiespossess a TENB2-binding arm and an arm which binds the cytotoxic agent(e.g., saporin, anti-interferon-α, vinca alkaloid, ricin A chain,methotrexate or radioactive isotope hapten). Bispecific antibodies canbe prepared as full length antibodies or antibody fragments (e.g.,F(ab′)₂ bispecific antibodies). Traditional production of full lengthbispecific antibodies is based on the co-expression of twoimmunoglobulin heavy chain-light chain pairs, where the two chains havedifferent specificities (Millstein et al (1983) Nature 305:537-539).

Heteroconjugate anti-TENB2 antibodies are also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells (U.S. Pat. No.4,676,980), and for treatment of HIV infection (WO 91/00360; WO92/200373; EP 03089). It is contemplated that the antibodies may beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents.

The anti-TENB2 antibodies of the present invention can be multivalentantibodies with three or more antigen binding sites (e.g. tetravalentantibodies), which can be readily produced by recombinant expression ofnucleic acid encoding the polypeptide chains of the antibody. Themultivalent antibody can comprise a dimerization domain and three ormore antigen binding sites. The preferred dimerization domain comprises(or consists of) an Fc region or a hinge region. In this scenario, theantibody will comprise an Fc region and three or more antigen bindingsites amino-terminal to the Fc region. The preferred multivalentantibody herein comprises (or consists of) three to about eight, butpreferably four, antigen binding sites. The multivalent antibodycomprises at least one polypeptide chain (and preferably two polypeptidechains), wherein the polypeptide chain(s) comprise two or more variabledomains. For instance, the polypeptide chain(s) may compriseVD1-(X1)_(n)-VD2-(X2)_(n)-Fc, wherein VD1 is a first variable domain,VD2 is a second variable domain, Fc is one polypeptide chain of an Fcregion, X1 and X2 represent an amino acid or polypeptide, and n is 0or 1. For instance, the polypeptide chain(s) may comprise:VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fcregion chain. The multivalent antibody herein preferably furthercomprises at least two (and preferably four) light chain variable domainpolypeptides. The multivalent antibody herein may, for instance,comprise from about two to about eight light chain variable domainpolypeptides. The light chain variable domain polypeptides contemplatedhere comprise a light chain variable domain and, optionally, furthercomprise a CL domain.

The effector function of an anti-TENB2 antibody may be modified byintroducing one or more amino acid substitutions in an Fc region. Suchmodification may enhance antigen-dependent cell-mediated cyotoxicity(ADCC) and/or complement dependent cytotoxicity (CDC) of the anti-TENB2antibody. The homodimeric antibody thus generated may have improvedinternalization capability and/or increased complement-mediated cellkilling and antibody-dependent cellular cytotoxicity (ADCC). See Caronet al (1992) J. Exp Med. 176:1191-1195 and Shopes, B. J. (1992) Immunol.148:2918-2922. Homodimeric anti-TENB2 antibodies with enhancedanti-tumor activity may also be prepared using heterobifunctionalcross-linkers as described in Wolff et al (1993) Cancer Research53:2560-2565. Alternatively, an antibody can be engineered which hasdual Fc regions and may thereby have enhanced complement lysis and ADCCcapabilities (Stevenson et al (1989) Anti-Cancer Drug Design 3:219-230).

The serum half life of an anti-TENB2 antibody may be modulated byincorporating a salvage receptor binding epitope, e.g. an antibodyfragment (U.S. Pat. No. 5,739,277). As used herein, the term “salvagereceptor binding epitope” refers to an epitope of the Fc region of anIgG molecule (e.g., IgG₁, IgG₂, IgG₃, or IgG₄) that is responsible forincreasing the in vivo serum half-life of the IgG molecule.

Monoclonal antibodies binding to TENB2 epitopes, including TMEFF2#19,are determined by standard competitive binding analysis and epitopemapping (PCT/US03/07209; U.S. Pat. No. 7,288,248).

Immunohistochemistry analysis was performed using TMEFF2#19 monoclonalantibodies (PCT/US03/07209; Sambrook et al Molecular Cloning: ALaboratory Manual, New York: Cold Spring Harbor Press, 1989; Ausubel etal., Current Protocols of Molecular Biology, Unit 3.16, John Wiley andSons, 1997). Monoclonal antibody TMEFF2#19 demonstarted weak to strongbinding in 176 of 241 human prostate cancer specimans.

Monoclonal antibody TMEFF2#19 becomes internalized into cells to whichit binds TENB2 polypeptide on the cell surface at a rapid rate.

Modifications of Anti-TENB2 Antibodies

Modifications and variations in the anti-TENB2 antibodies describedherein, can be made, for example, using any of the techniques andguidelines known in the art for conservative and non-conservativemutations, for example, those in U.S. Pat. No. 5,364,934. Variations maybe a substitution, deletion or insertion of one or more codons encodingthe antibody or polypeptide that results in a change in the amino acidsequence as compared with the native sequence anti-TENB2 antibody.Optionally the variation is by substitution of at least one amino acidwith any other amino acid in one or more of the domains of theanti-TENB2 antibody. The variations can be made using methods known inthe art such as oligonucleotide-mediated (site-directed) mutagenesis,alanine scanning, and PCR mutagenesis. Site-directed mutagenesis (Carteret al (1986) Nucl. Acids Res., 13:4331; Zoller et al (1987) Nucl. AcidsRes., 10:6487), cassette mutagenesis (Wells et al (1985) Gene, 34:315),restriction selection mutagenesis (Wells et al (1986) Philos. Trans. R.Soc. London SerA, 317:415) or other known techniques can be performed onthe cloned DNA to produce the anti-TENB2 antibody variant DNA. Aminoacid changes may alter post-translational processes of the anti-TENB2antibody, such as changing the number or position of glycosylation sitesor altering the membrane anchoring characteristics. Other modificationsinclude deamidation of glutaminyl and asparaginyl residues to thecorresponding glutamyl and aspartyl residues, respectively,hydroxylation of proline and lysine, phosphorylation of hydroxyl groupsof seryl or threonyl residues, methylation of the α-amino groups oflysine, arginine, and histidine side chains (T. E. Creighton, Proteins:Structure and Molecular Properties, (1983) W.H. Freeman & Co., SanFrancisco, pp. 79-86), acetylation of the N-terminal amine, andamidation of any C-terminal carboxyl group. Anti-TENB2 antibodies can beprepared by introducing appropriate nucleotide changes into the encodingDNA, and/or by chemical synthesis.

Anti-TENB2 antibody fragments may be truncated at the N-terminus orC-terminus, or may lack internal residues, for example, when comparedwith a full length anti-TENB2 antibody. Certain fragments lack aminoacid residues that are not essential for a desired biological activityof the anti-TENB2 antibody. Anti-TENB2 antibody fragments may beprepared by any of a number of conventional techniques. Desired peptidefragments may be chemically synthesized. An alternative approachinvolves generating antibody fragments by enzymatic digestion, e.g., bytreating the protein with an enzyme known to cleave proteins at sitesdefined by particular amino acid residues, or by digesting the DNA withsuitable restriction enzymes and isolating the desired fragment. Yetanother suitable technique involves isolating and amplifying a DNAfragment encoding a desired antibody or fragment, by polymerase chainreaction (PCR). Oligonucleotides that define the desired termini of theDNA fragment are employed at the 5′ and 3′ primers in the PCR.Preferably, anti-TENB2 antibody fragments share at least one biologicaland/or immunological activity with the native anti-TENB2 antibodydisclosed herein.

A particularly preferred type of substitutional variant involvessubstituting one or more hypervariable region residues of a humanized orhuman antibody. Generally, the resulting variant(s) selected for furtherdevelopment will have improved biological properties relative to theantibody from which they are generated. A convenient way for generatingsuch substitutional variants involves affinity maturation using phagedisplay. Briefly, several hypervariable region sites (e.g., 6-7 sites)are mutated to generate all possible amino substitutions at each site.The antibody variants thus generated are displayed in a monovalentfashion from filamentous phage particles as fusions to the gene IIIproduct of M13 packaged within each particle. The phage-displayedvariants are then screened for their biological activity (e.g., bindingaffinity) as herein disclosed. In order to identify candidatehypervariable region sites for modification, alanine scanningmutagenesis can be performed to identify hypervariable region residuescontributing significantly to antigen binding. Alternatively, oradditionally, it may be beneficial to analyze a crystal structure of theantigen-antibody complex to identify contact points between the antibodyand human TENB2 polypeptide. Such contact residues and neighboringresidues are candidates for substitution according to the techniqueselaborated herein. Once such variants are generated, the panel ofvariants is subjected to screening as described herein and antibodieswith superior properties in one or more relevant assays may be selectedfor further development.

Another type of covalent modification of the anti-TENB2 antibodyincluded within the scope of this invention comprises altering thenative glycosylation pattern of the antibody or polypeptide by deletingone or more carbohydrate moieties found in native sequence anti-TENB2antibody (either by removing the underlying glycosylation site or bydeleting the glycosylation by chemical and/or enzymatic means), and/oradding one or more glycosylation sites that are not present in thenative sequence anti-TENB2 antibody. In addition, the modificationincludes qualitative changes in the glycosylation of the nativeproteins, involving a change in the nature and proportions of thevarious carbohydrate moieties present. Glycosylation of antibodies andother polypeptides is typically either N-linked or O-linked. N-linkedrefers to the attachment of the carbohydrate moiety to the side chain ofan asparagine residue. The tripeptide sequences asparagine-X-serine andasparagine-X-threonine, where X is any amino acid except proline, arethe recognition sequences for enzymatic attachment of the carbohydratemoiety to the asparagine side chain. Thus, the presence of either ofthese tripeptide sequences in a polypeptide creates a potentialglycosylation site. O-Linked glycosylation refers to the attachment ofone of the sugars N-acetylgalactosamine, galactose, or xylose to ahydroxyamino acid, most commonly serine or threonine, although5-hydroxyproline or 5-hydroxylysine may also be used. Addition ofglycosylation sites to the anti-TENB2 antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tripeptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thesequence of the anti-TENB2 antibody (for O-linked glycosylation sites).The anti-TENB2 antibody amino acid sequence may optionally be alteredthrough changes at the DNA level, particularly by mutating the DNAencoding the anti-TENB2 antibody at preselected bases such that codonsare generated that will translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theanti-TENB2 antibody is by chemical or enzymatic coupling of glycosidesto the polypeptide. Such methods are described in the art, e.g., in WO87/05330 published 11 Sep. 1987, and in Aplin and Wriston, CRC Crit.Rev. Biochem., pp. 259-306 (1981).

Removal of carbohydrate moieties present on the anti-TENB2 antibody maybe accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties can beachieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al (1987) Meth. Enzymol. 138:350.

Another type of covalent modification of anti-TENB2 antibody compriseslinking the antibody or polypeptide to one of a variety ofnonproteinaceous polymers, e.g., polyethylene glycol (PEG),polypropylene glycol, or polyoxyalkylenes, in the manner set forth inU.S. Pat. No. 4,640,835; U.S. Pat. No. 4,496,689; U.S. Pat. No.4,301,144; U.S. Pat. No. 4,670,417; U.S. Pat. No. 4,791,192 or U.S. Pat.No. 4,179,337. The antibody or polypeptide also may be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization (for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively), in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules), or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, 16th edition, Oslo, A., Ed.,(1980).

The anti-TENB2 antibody of the present invention may also be modified ina way to form chimeric molecules comprising an anti-TENB2 antibody fusedto another, heterologous polypeptide or amino acid sequence. In oneembodiment, such a chimeric molecule comprises a fusion of theanti-TENB2 antibody with a tag polypeptide which provides an epitope towhich an anti-tag antibody can selectively bind. The epitope tag isgenerally placed at the amino- or carboxyl-terminus of the anti-TENB2antibody. The presence of such epitope-tagged forms of the anti-TENB2antibody can be detected using an antibody against the tag polypeptide.Also, provision of the epitope tag enables the anti-TENB2 antibody to bereadily purified by affinity purification using an anti-tag antibody oranother type of affinity matrix that binds to the epitope tag. Varioustag polypeptides and their respective antibodies are well known in theart. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 (Field et al (1988) Mol. Cell. Biol.,8:2159-2165); the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto (Evan et al (1985) Molecular and Cellular Biology,5:3610-3616); and the Herpes Simplex virus glycoprotein D (gD) tag andits antibody (Paborsky et al (1990) Protein Engineering, 3(6):547-553).Other tag polypeptides include the Flag-peptide (Hopp et al (1988)BioTechnology 6:1204-1210); the KT3 epitope peptide (Martin et al (1992)Science, 255:192-194); an α-tubulin epitope peptide (Skinner et al(1991) J. Biol. Chem., 266:15163-15166); and the T7 gene 10 proteinpeptide tag (Lutz-Freyermuth et al (1990) Proc. Natl. Acad. Sci. USA,87:6393-6397).

In an alternative embodiment, the chimeric molecule may comprise afusion of the anti-TENB2 antibody with an immunoglobulin or a particularregion of an immunoglobulin. For a bivalent form of the chimericmolecule (also referred to as an “immunoadhesin”), such a fusion couldbe to the Fc region of an IgG molecule. The Ig fusions preferablyinclude the substitution of a soluble (transmembrane domain deleted orinactivated) form of an anti-TENB2 antibody in place of at least onevariable region within an Ig molecule. In a particularly preferredembodiment, the immunoglobulin fusion includes the hinge, CH₂ and CH₃,or the hinge, CH₁, CH₂ and CH₃ regions of an IgG1 molecule (U.S. Pat.No. 5,428,130).

Preparation of Anti-TENB2 Antibodies

DNA encoding an amino acid sequence variant of the cysteine engineeredanti-TENB2 antibodies and parent anti-TENB2 antibodies of the inventionis prepared by a variety of methods which include, but are not limitedto, isolation from a natural source (in the case of naturally occurringamino acid sequence variants), preparation by site-directed (oroligonucleotide-mediated) mutagenesis (Carter (1985) et al Nucleic AcidsRes. 13:4431-4443; Ho et al (1989) Gene (Amst.) 77:51-59; Kunkel et al(1987) Proc. Natl. Acad. Sci. USA 82:488; Liu et al (1998) J. Biol.Chem. 273:20252-20260), PCR mutagenesis (Higuchi, (1990) in PCRProtocols, pp. 177-183, Academic Press; Ito et al (1991) Gene 102:67-70;Bernhard et al (1994) Bioconjugate Chem. 5:126-132; and Vallette et al(1989) Nuc. Acids Res. 17:723-733), and cassette mutagenesis (Wells etal (1985) Gene 34:315-323) of an earlier prepared DNA encoding thepolypeptide. Mutagenesis protocols, kits, and reagents are commerciallyavailable, e.g. QuikChange® Multi Site-Direct Mutagenesis Kit(Stratagene, La Jolla, Calif.). Single mutations are also generated byoligonucleotide directed mutagenesis using double stranded plasmid DNAas template by PCR based mutagenesis (Sambrook and Russel, (2001)Molecular Cloning: A Laboratory Manual, 3rd edition; Zoller et al (1983)Methods Enzymol. 100:468-500; Zoller, M. J. and Smith, M. (1982) Nucl.Acids Res. 10:6487-6500). Variants of recombinant antibodies may beconstructed also by restriction fragment manipulation or by overlapextension PCR with synthetic oligonucleotides. Mutagenic primers encodethe cysteine codon replacement(s). Standard mutagenesis techniques canbe employed to generate DNA encoding such mutant cysteine engineeredantibodies (Sambrook et al Molecular Cloning, A Laboratory Manual, ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989; andAusubel et al Current Protocols in Molecular Biology, Greene Publishingand Wiley-Interscience, New York, N.Y., 1993).

Phage display technology (McCafferty et al (1990) Nature 348:552-553)can be used to produce anti-TENB2 human antibodies and antibodyfragments in vitro, from immunoglobulin variable (V) domain generepertoires from unimmunized donors. According to this technique,antibody V domain genes are cloned in-frame into either a major or minorcoat protein gene of a filamentous bacteriophage, such as M13 or fd, anddisplayed as functional antibody fragments on the surface of the phageparticle. Because the filamentous particle contains a single-strandedDNA copy of the phage genome, selections based on the functionalproperties of the antibody also result in selection of the gene encodingthe antibody exhibiting those properties. Thus, the phage mimics some ofthe properties of the B-cell (Johnson et al (1993) Current Opinion inStructural Biology 3:564-571; Clackson et al (1991) Nature, 352:624-628;Marks et al (1991) J. Mol. Biol. 222:581-597; Griffith et al (1993) EMBOJ. 12:725-734; U.S. Pat. No. 5,565,332; U.S. Pat. No. 5,573,905; U.S.Pat. No. 5,567,610; U.S. Pat. No. 5,229,275).

Anti-TENB2 antibodies may be chemically synthesized using knownoligopeptide synthesis methodology or may be prepared and purified usingrecombinant technology. The appropriate amino acid sequence, or portionsthereof, may be produced by direct peptide synthesis using solid-phasetechniques (Stewart et al., Solid-Phase Peptide Synthesis, (1969) W.H.Freeman Co., San Francisco, Calif.; Merrifield, (1963) J. Am. Chem.Soc., 85:2149-2154). In vitro protein synthesis may be performed usingmanual techniques or by automation. Automated solid phase synthesis maybe accomplished, for instance, employing t-BOC or Fmoc protected aminoacids and using an Applied Biosystems Peptide Synthesizer (Foster City,Calif.) using manufacturer's instructions. Various portions of theanti-TENB2 antibody or TENB2 polypeptide may be chemically synthesizedseparately and combined using chemical or enzymatic methods to producethe desired anti-TENB2 antibody or TENB2 polypeptide.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (Morimoto et al (1992) Journal ofBiochemical and Biophysical Methods 24:107-117; and Brennan et al (1985)Science, 229:81), or produced directly by recombinant host cells. Fab,Fv and ScFv anti-TENB2 antibody fragments can all be expressed in andsecreted from E. coli, thus allowing the facile production of largeamounts of these fragments. Antibody fragments can be isolated from theantibody phage libraries discussed herein. Alternatively, Fab′-SHfragments can be directly recovered from E. coli and chemically coupledto form F(ab′)₂ fragments (Carter et al (1992) Bio/Technology10:163-167), or isolated directly from recombinant host cell culture.The anti-TENB2 antibody may be a (scFv) single chain Fv fragment (WO93/16185; U.S. Pat. No. 5,571,894; U.S. Pat. No. 5,587,458). Theanti-TENB2 antibody fragment may also be a “linear antibody” (U.S. Pat.No. 5,641,870). Such linear antibody fragments may be monospecific orbispecific.

The description below relates primarily to production of anti-TENB2antibodies by culturing cells transformed or transfected with a vectorcontaining anti-TENB2 antibody-encoding nucleic acid. DNA encodinganti-TENB2 antibodies may be obtained from a cDNA library prepared fromtissue believed to possess the anti-TENB2 antibody mRNA and to expressit at a detectable level. Accordingly, human anti-TENB2 antibody orTENB2 polypeptide DNA can be conveniently obtained from a cDNA libraryprepared from human tissue. The anti-TENB2 antibody-encoding gene mayalso be obtained from a genomic library or by known synthetic procedures(e.g., automated nucleic acid synthesis).

Libraries can be screened with probes (such as oligonucleotides of atleast about 20-80 bases) designed to identify the gene of interest orthe protein encoded by it. Screening the cDNA or genomic library withthe selected probe may be conducted using standard procedures, such asdescribed in Sambrook et al., Molecular Cloning: A Laboratory Manual(New York: Cold Spring Harbor Laboratory Press, 1989). An alternativemeans to isolate the gene encoding anti-TENB2 antibody or TENB2polypeptide is PCR methodology (Sambrook et al., supra; Dieffenbach etal., PCR Primer: A Laboratory Manual, Cold Spring Harbor LaboratoryPress, 1995).

Host cells are transfected or transformed with expression or cloningvectors described herein for anti-TENB2 antibody or TENB2 polypeptideproduction and cultured in conventional nutrient media modified asappropriate for inducing promoters, selecting transformants, oramplifying the genes encoding the desired sequences. The cultureconditions, such as media, temperature, pH and the like, can be selectedby the skilled artisan without undue experimentation. In general,principles, protocols, and practical techniques for maximizing theproductivity of cell cultures can be found in Mammalian CellBiotechnology: a Practical Approach, M. Butler, ed. (IRL Press, 1991)and Sambrook et al., supra.

Suitable host cells for cloning or expressing the DNA in the vectorsherein include prokaryote, yeast, or higher eukaryote cells. Suitableprokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E. coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776(ATCC 31,537); E. coli strain W3110 (ATCC 27,325) and K5 772 (ATCC53,635). Other suitable prokaryotic host cells includeEnterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. These examples are illustrative ratherthan limiting. Strain W3110 is an exemplary host strain for recombinantDNA product fermentations. Preferably, the host cell secretes minimalamounts of proteolytic enzymes. For example, strain W3110 may bemodified to effect a genetic mutation in the genes encoding proteinsendogenous to the host, with examples of such hosts including E. coliW3110 strain 1A2, which has the complete genotype tonA; E. coli W3110strain 9E4, which has the complete genotype tonA ptr3; E. coli W3110strain 27C7 (ATCC 55,244), which has the complete genotype tonA ptr3phoA E15 (argF-lac)169 degP ompT kan^(r) ; E. coli W31 10 strain 37D6,which has the complete genotype tonA ptr3 phoA E15 (argF-lac)169 degPompT rbs7 ilvG kan^(r) ; E. coli W3110 strain 40B4, which is strain 37D6with a non-kanamycin resistant degP deletion mutation; and an E. colistrain having mutant periplasmic protease (U.S. Pat. No. 4,946,783).Alternatively, in vitro methods of cloning, e.g., PCR or other nucleicacid polymerase reactions, are suitable.

Full length antibody, antibody fragments, and antibody fusion proteinscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) and theimmunoconjugate by itself shows effectiveness in tumor cell destruction.Full length antibodies have greater half life in circulation. Productionin E. coli may be faster and more cost efficient using, for example,expression of antibody fragments and polypeptides in bacteria withtranslation initiation regio (TIR) and signal sequences for optimizingexpression and secretion (U.S. Pat. No. 5,648,237; U.S. Pat. No.5,789,199; U.S. Pat. No. 5,840,523). After expression, the antibody isisolated from the E. coli cell paste in a soluble fraction and can bepurified through, e.g., a protein A or G column depending on theisotype. Final purification can be carried out similar to the processfor purifying antibody expressed e.g, in CHO cells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts for anti-TENB2antibody- or TENB2 polypeptide-encoding vectors. Saccharomycescerevisiae is a commonly used lower eukaryotic host microorganism.Others include Schizosaccharomyces pombe (Beach and Nurse, (1981)Nature, 290: 140; EP 139,383); Kluyveromyces hosts (U.S. Pat. No.4,943,529; Fleer et al (1991) Bio/Technology, 9:968-975) such as, e.g.,K. lactis (MW98-8C, CBS683, CBS4574; Louvencourt et al (1983) J.Bacteriol., 154(2):737-742), K. fragilis (ATCC 12,424), K. bulgaricus(ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K.drosophilarum (ATCC 36,906; Van den Berg et al (1990) Bio/Technology,8:135), K. thermotolerans, and K. marxianus; yarrowia (EP 402226);Pichia pastoris (EP 183070; Sreekrishna et al (1988) J. BasicMicrobiol., 28:265-278); Candida; Trichoderma reesia (EP 244234);Neurospora crassa(Case et al (1979) Proc. Natl. Acad. Sci. USA,76:5259-5263); Schwanniomyces such as Schwanniomyces occidentalis (EP394538); and filamentous fungi such as, e.g., Neurospora, Penicillium,Tolypocladium (WO 91/00357), and Aspergillus hosts such as A. nidulans(Ballance et al (1983) Biochem. Biophys. Res. Commun., 112:284-289;Tilburn et al (1983) Gene, 26:205-221; Yelton et al (1984) Proc. Natl.Acad. Sci. USA, 81: 1470-1474) and A. niger (Kelly and Hynes, (1985)EMBO J., 4:475-479). Methylotropic yeasts are suitable herein andinclude, but are not limited to, yeast capable of growth on methanolselected from the genera consisting of Hansenula, Candida, Kloeckera,Pichia, Saccharomyces, Torulopsis, and Rhodotorula.

Suitable host cells for the expression of glycosylated anti-TENB2antibody or TENB2 polypeptide may also be derived from multicellularorganisms. Examples of invertebrate cells include insect cells such asDrosophila S2 and Spodoptera Sf9, as well as plant cells, such as cellcultures of cotton, corn, potato, soybean, petunia, tomato, and tobacco.Numerous baculoviral strains and variants and corresponding permissiveinsect host cells from hosts such as Spodoptera frugiperda(caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito),Drosophila melanogaster (fruitfly), and Bombyx mori have beenidentified. A variety of viral strains for transfection are publiclyavailable, e.g., the L-1 variant of Autographa californica NPV and theBm-5 strain of Bombyx mori NPV, and such viruses may be used as thevirus herein according to the present invention, particularly fortransfection of Spodoptera frugiperda cells.

Examples of useful mammalian host cell lines are monkey kidney CV 1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal (1977) J. Gen Virol. 36:59); baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells/-DHFR (CHO, Urlaub et al (1980) Proc.Natl. Acad. Sci. USA 77:4216); mouse sertoli cells (TM4, Mather (1980)Biol. Reprod. 23:243-251); monkey kidney cells (CV 1 ATCC CCL 70);African green monkey kidney cells (VERO-76, ATCC CRL-1587); humancervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK,ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); humanlung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065);mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al(1982) Annals N.Y. Acad. Sci. 383:44-68); MRC 5 cells; FS4 cells; and ahuman hepatoma line (Hep G2).

Host cells are transformed with the above-described expression orcloning vectors for anti-TENB2 antibody production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The nucleic acid (e.g., cDNA or genomic DNA) encodinganti-TENB2 antibody or TENB2 polypeptide may be inserted into areplicable vector for cloning (amplification of the DNA) or forexpression. The vector may, for example, be in the form of a plasmid,cosmid, viral particle, or phage. The appropriate nucleic acid sequencemay be inserted into the vector by a variety of procedures.

Growth inhibition of tumor cells in vitro or in vivo can be determinedin various ways known in the art, such as inhibiting cell proliferationof a TENB2-expressing tumor cell in vitro or in vivo by about 25-100%compared to the untreated tumor cell, or by about 30-100%, or by about50-100% or 70-100%, in one embodiment, at an antibody concentration ofabout 0.5 to 30 gig/ml. The growth inhibitory effects of an anti-TENB2antibody in vitro may be assessed by methods known in the art, e.g.,using cells which express a TENB2 polypeptide either endogenously orfollowing transfection with the TENB2 gene. For example, appropriatetumor cell lines and TENB2-transfected cells may treated with ananti-TENB2 monoclonal antibody at various concentrations for a few days(e.g., 2-7) days and stained with crystal violet or MTT or analyzed bysome other colorimetric assay. A reduced signal indicates growthinhibition. Another method of measuring proliferation would be bycomparing ³H-thymidine uptake by the cells treated in the presence orabsence an anti-TENB2 antibody. After treatment, the cells are harvestedand the amount of radioactivity incorporated into the DNA quantitated ina scintillation counter. Inhibition of proliferation would bedemonstrated by a reduction of radioactivity. To select for ananti-TENB2 antibody which induces cell death, loss of membrane integrityas indicated by, e.g., propidium iodide (PI), trypan blue or 7AADuptake, may be assessed relative to control. Appropriate positivecontrols include treatment of a selected cell line with a growthinhibitory antibody known to inhibit growth of that cell line. Growthinhibition can be measured at an antibody concentration of about 0.5 to30 μg/ml or about 0.5 nM to 200 nM in cell culture, where the growthinhibition is determined 1-10 days after exposure of the tumor cells tothe antibody. The antibody is growth inhibitory in vivo ifadministration of the anti-TENB2 antibody at about 1 gig/kg to about 100mg/kg body weight results in reduction in tumor size or reduction oftumor cell proliferation within about 5 days to 3 months from the firstadministration of the antibody, preferably within about 5 to 30 days.

Preparation of Cysteine Engineered Anti-TENB2 Antibodies

The design, selection, and preparation methods of the invention enablecysteine engineered anti-TENB2 antibodies which are reactive withelectrophilic functionality. These methods further enable antibodyconjugate compounds such as antibody-drug conjugate (ADC) compounds withdrug molecules at designated, designed, selective sites. Reactivecysteine residues on an antibody surface allow specifically conjugatinga drug moiety through a thiol reactive group such as maleimide orhaloacetyl. The nucleophilic reactivity of the thiol functionality of aCys residue to a maleimide group is about 1000 times higher compared toany other amino acid functionality in a protein, such as amino group oflysine residues or the N-terminal amino group. Thiol specificfunctionality in iodoacetyl and maleimide reagents may react with aminegroups, but higher pH (>9.0) and longer reaction times are required(Garman, 1997, Non-Radioactive Labelling: A Practical Approach, AcademicPress, London). The amount of free thiol in a protein may be estimatedby the standard Ellman's assay. Immunoglobulin M is an example of adisulfide-linked pentamer, while immunoglobulin G is an example of aprotein with internal disulfide bridges bonding the subunits together.In proteins such as this, reduction of the disulfide bonds with areagent such as dithiothreitol (DTT) or selenol (Singh et al (2002)Anal. Biochem. 304:147-156) is required to generate the reactive freethiol. This approach may result in loss of antibody tertiary structureand antigen binding specificity.

The PHESELECTOR (Phage ELISA for Selection of Reactive Thiols) Assayallows for detection of reactive cysteine groups in antibody-Fabs in anELISA phage format thereby assisting in the design of cysteineengineered antibodies (US 2007/0092940). The antigen that binds tocysteine engineered antibody is coated on well surfaces, followed byincubation with phage particles displaying cysteine engineered Fabs,addition of HRP labeled secondary antibody, and absorbance detection.Mutant proteins displayed on phage may be screened in a rapid, robust,and high-throughput manner. Libraries of cysteine engineered antibodiescan be produced and subjected to binding selection using the sameapproach to identify appropriately reactive sites of free Cysincorporation from random protein-phage libraries of antibodies or otherproteins. This technique includes reacting cysteine mutant proteinsdisplayed on phage with an affinity reagent or reporter group which isalso thiol-reactive.

The PHESELECTOR assay allows screening of reactive thiol groups inantibodies. Identification of the A121C variant by this method isexemplary. The entire Fab molecule may be effectively searched toidentify more ThioFab variants with reactive thiol groups. A parameter,fractional surface accessibility, was employed to identify andquantitate the accessibility of solvent to the amino acid residues in apolypeptide. The surface accessibility can be expressed as the surfacearea (Å²) that can be contacted by a solvent molecule, e.g. water. Theoccupied space of water is approximated as a 1.4 A radius sphere.Software is freely available or licensable (Secretary to CCP4, DaresburyLaboratory, Warrington, WA4 4AD, United Kingdom, Fax: (+44) 1925 603825)as the CCP4 Suite of crystallography programs which employ algorithms tocalculate the surface accessibility of each amino acid of a protein withknown x-ray crystallography derived coordinates (“The CCP4 Suite:Programs for Protein Crystallography” (1994) Acta. Cryst. D50:760-763).Two exemplary software modules that perform surface accessibilitycalculations are “AREAIMOL” and “SURFACE”, based on the algorithms of B.Lee and F. M. Richards (1971) J. Mol. Biol. 55:379-400. AREAIMOL definesthe solvent accessible surface of a protein as the locus of the centreof a probe sphere (representing a solvent molecule) as it rolls over theVan der Waals surface of the protein. AREAIMOL calculates the solventaccessible surface area by generating surface points on an extendedsphere about each atom (at a distance from the atom centre equal to thesum of the atom and probe radii), and eliminating those that lie withinequivalent spheres associated with neighboring atoms. AREAIMOL finds thesolvent accessible area of atoms in a PDB coordinate file, andsummarizes the accessible area by residue, by chain and for the wholemolecule. Accessible areas (or area differences) for individual atomscan be written to a pseudo-PDB output file. AREAIMOL assumes a singleradius for each element, and only recognizes a limited number ofdifferent elements.

AREAIMOL and SURFACE report absolute accessibilities, i.e. the number ofsquare Angstroms (A). Fractional surface accessibility is calculated byreference to a standard state relevant for an amino acid within apolypeptide. The reference state is tripeptide Gly-X-Gly, where X is theamino acid of interest, and the reference state should be an ‘extended’conformation, i.e. like those in beta-strands. The extended conformationmaximizes the accessibility of X. A calculated accessible area isdivided by the accessible area in a Gly-X-Gly tripeptide reference stateand reports the quotient, which is the fractional accessibility. Percentaccessibility is fractional accessibility multiplied by 100. Anotherexemplary algorithm for calculating surface accessibility is based onthe SOLV module of the program xsae (Broger, C., F. Hoffman-LaRoche,Basel) which calculates fractional accessibility of an amino acidresidue to a water sphere based on the X-ray coordinates of thepolypeptide. The fractional surface accessibility for every amino acidin an antibody may be calculated using available crystal structureinformation (Eigenbrot et al. (1993) J Mol Biol. 229:969-995).

DNA encoding the cysteine engineered antibodies is readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). The hybridoma cells serveas a source of such DNA. Once isolated, the DNA may be placed intoexpression vectors, which are then transfected into host cells such asE. coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, orother mammalian host cells, such as myeloma cells (U.S. Pat. No.5,807,715; US 2005/0048572; US 2004/0229310) that do not otherwiseproduce the antibody protein, to obtain the synthesis of monoclonalantibodies in the recombinant host cells.

After design and selection, cysteine engineered antibodies, e.g.ThioFabs, with the engineered, highly reactive unpaired Cys residues,may be produced by: (i) expression in a bacterial, e.g. E. coli, system(Skerra et al (1993) Curr. Opinion in Immunol. 5:256-262; Plückthun(1992) Immunol. Revs. 130:151-188) or a mammalian cell culture system(WO 01/00245), e.g. Chinese Hamster Ovary cells (CHO); and (ii)purification using common protein purification techniques (Lowman et al(1991) J. Biol. Chem. 266(17): 10982-10988).

The engineered Cys thiol groups react with electrophilic linker reagentsand drug-linker intermediates to form cysteine engineered antibody drugconjugates and other labelled cysteine engineered antibodies. Cysresidues of cysteine engineered antibodies, and present in the parentantibodies, which are paired and form interchain and intrachaindisulfide bonds do not have any reactive thiol groups (unless treatedwith a reducing agent) and do not react with electrophilic linkerreagents or drug-linker intermediates. The newly engineered Cys residue,can remain unpaired, and able to react with, i.e. conjugate to, anelectrophilic linker reagent or drug-linker intermediate, such as adrug-maleimide. Exemplary drug-linker intermediates include: MC-MMAE,MC-MMAF, MC-vc-PAB-MMAE, and MC-vc-PAB-MMAF. The structure positions ofthe engineered Cys residues of the heavy and light chains are numberedaccording to a sequential numbering system. This sequential numberingsystem is correlated to the Kabat numbering system (Kabat et al., (1991)Sequences of Proteins of Immunological Interest, 5th Ed. Public HealthService, National Institutes of Health, Bethesda, Md.) starting at theN-terminus, differs from the Kabat numbering scheme (bottom row) byinsertions noted by a,b,c. Using the Kabat numbering system, the actuallinear amino acid sequence may contain fewer or additional amino acidscorresponding to a shortening of, or insertion into, a FR or CDR of thevariable domain. The cysteine engineered heavy chain variant sites areidentified by the sequential numbering and Kabat numbering schemes.

In one embodiment, the cysteine engineered anti-TENB2 antibody isprepared by a process comprising:

(a) replacing one or more amino acid residues of a parent anti-TENB2antibody by cysteine; and

(b) determining the thiol reactivity of the cysteine engineeredanti-TENB2 antibody by reacting the cysteine engineered antibody with athiol-reactive reagent.

The cysteine engineered antibody may be more reactive than the parentantibody with the thiol-reactive reagent.

The free cysteine amino acid residues may be located in the heavy orlight chains, or in the constant or variable domains. Antibodyfragments, e.g. Fab, may also be engineered with one or more cysteineamino acids replacing amino acids of the antibody fragment, to formcysteine engineered antibody fragments.

Another embodiment of the invention provides a method of preparing(making) a cysteine engineered anti-TENB2 antibody, comprising:

(a) introducing one or more cysteine amino acids into a parentanti-TENB2 antibody in order to generate the cysteine engineeredanti-TENB2 antibody; and

(b) determining the thiol reactivity of the cysteine engineered antibodywith a thiol-reactive reagent;

-   -   wherein the cysteine engineered antibody is more reactive than        the parent antibody with the thiol-reactive reagent.

Step (a) of the method of preparing a cysteine engineered antibody maycomprise:

(i) mutagenizing a nucleic acid sequence encoding the cysteineengineered antibody;

(ii) expressing the cysteine engineered antibody; and

(iii) isolating and purifying the cysteine engineered antibody.

Step (b) of the method of preparing a cysteine engineered antibody maycomprise expressing the cysteine engineered antibody on a viral particleselected from a phage or a phagemid particle.

Step (b) of the method of preparing a cysteine engineered antibody mayalso comprise:

(i) reacting the cysteine engineered antibody with a thiol-reactiveaffinity reagent to generate an affinity labelled, cysteine engineeredantibody; and

(ii) measuring the binding of the affinity labelled, cysteine engineeredantibody to a capture media.

Another embodiment of the invention is a method of screening cysteineengineered antibodies with highly reactive, unpaired cysteine aminoacids for thiol reactivity comprising:

(a) introducing one or more cysteine amino acids into a parent antibodyin order to generate a cysteine engineered antibody;

(b) reacting the cysteine engineered antibody with a thiol-reactiveaffinity reagent to generate an affinity labelled, cysteine engineeredantibody; and

(c) measuring the binding of the affinity labelled, cysteine engineeredantibody to a capture media; and

(d) determining the thiol reactivity of the cysteine engineered antibodywith the thiol-reactive reagent.

Step (a) of the method of screening cysteine engineered antibodies maycomprise:

(i) mutagenizing a nucleic acid sequence encoding the cysteineengineered antibody;

(ii) expressing the cysteine engineered antibody; and

(iii) isolating and purifying the cysteine engineered antibody.

Step (b) of the method of screening cysteine engineered antibodies maycomprise expressing the cysteine engineered antibody on a viral particleselected from a phage or a phagemid particle.

Step (b) of the method of screening cysteine engineered antibodies mayalso comprise:

(i) reacting the cysteine engineered antibody with a thiol-reactiveaffinity reagent to generate an affinity labelled, cysteine engineeredantibody; and

(ii) measuring the binding of the affinity labelled, cysteine engineeredantibody to a capture media.

Cysteine Engineering of TMEFF2#19 IgG Variants

Cysteine was introduced at the heavy chain 121 (sequential numberingexcluding the signal sequence) site into full-length, humanized parentmonoclonal anti-TENB2 TMEFF2#19 antibodies by the cysteine engineeringmethods described herein to give A121C thio hu anti-TENB2 TMEFF2#19humanized variant with heavy chain sequence: SEQ ID NO: 1, and lightchain sequence: SEQ ID NO:2, FIG. 1. These cysteine engineeredmonoclonal antibodies were expressed in CHO (Chinese Hamster Ovary)cells by transient fermentation in media containing 1 mM cysteine.

According to one embodiment, humanized TMEFF2#19 cysteine engineeredanti-TENB2 antibodies comprise one or more of the following variableregion heavy chain sequences with a free cysteine amino acid (Table 1).

TABLE 1 Comparison of heavy chain Sequential, Kabatand Eu numbering for hu TMEFF2 #19 cysteineengineered anti-TENB2 antibody variants Sequence Kabat Eu near CysSequential Num- Num- Seq mutation Numbering bering bering I.D.DVQLCESGPG Q5C Q5C 8 LSLTCCVSGYS A23C A23C 9 LSSVTCADTAV A88C A84C 10TLVTVCSASTK S119C S112C 11 VTVSSCSTKGP A121C A114C A118C 12 VSSASCKGPSVT123C T116C T120C 13 WYVDGCEVHNA V285C V278C V282C 14 KGFYPCDIAVE S378CS371C S375C 15 PPVLDCDGSFF 5403C S396C S400C 16

According to one embodiment, humanized TMEFF2#19 cysteine engineeredanti-TENB2 antibodies comprise one or more of the following variableregion light chain sequences with a free cysteine amino acid (Table 2).

TABLE 2 Comparison of light chain Sequential andKabat numbering for hu TMEFF2 #19 cysteineengineered anti-TENB2 antibody variants Sequence near Cys SequentialKabat Seq. mutation Numbering Numbering I.D. SLSASCGDRVT V15C V15C 17EIKRTCAAPSV V110C V110C 18 TVAAPCVFIFP S114C S114C 19 FIFPPCDEQLK S121CS121C 20 DEQLKCGTASV S127C S127C 21 VTEQDCKDSTY S168C S168C 22GLSSPCTKSFN V205C V205C 23

Labelled Cysteine Engineered Anti-TENB2 Antibodies

Cysteine engineered anti-TENB2 antibodies may be site-specifically andefficiently coupled with a thiol-reactive reagent. The thiol-reactivereagent may be a multifunctional linker reagent, a capture, i.e.affinity, label reagent (e.g. a biotin-linker reagent), a detectionlabel (e.g. a fluorophore reagent), a solid phase immobilization reagent(e.g. SEPHAROSE™, polystyrene, or glass), or a drug-linker intermediate.One example of a thiol-reactive reagent is N-ethyl maleimide (NEM). Inan exemplary embodiment, reaction of a ThioFab with a biotin-linkerreagent provides a biotinylated ThioFab by which the presence andreactivity of the engineered cysteine residue may be detected andmeasured. Reaction of a ThioFab with a multifunctional linker reagentprovides a ThioFab with a functionalized linker which may be furtherreacted with a drug moiety reagent or other label. Reaction of a ThioFabwith a drug-linker intermediate provides a ThioFab drug conjugate.

The exemplary methods described here may be applied generally to theidentification and production of antibodies, and more generally, toother proteins through application of the design and screening stepsdescribed herein.

Such an approach may be applied to the conjugation of otherthiol-reactive reagents in which the reactive group is, for example, amaleimide, an iodoacetamide, a pyridyl disulfide, or otherthiol-reactive conjugation partner (Haugland, 2003, Molecular ProbesHandbook of Fluorescent Probes and Research Chemicals, Molecular Probes,Inc.; Brinkley, 1992, Bioconjugate Chem. 3:2; Garman, 1997,Non-Radioactive Labelling: A Practical Approach, Academic Press, London;Means (1990) Bioconjugate Chem. 1:2; Hermanson, G. in BioconjugateTechniques (1996) Academic Press, San Diego, pp. 40-55, 643-671). Thethiol-reactive reagent may be a drug moiety, a fluorophore such as afluorescent dye like fluorescein or rhodamine, a chelating agent for animaging or radiotherapeutic metal, a peptidyl or non-peptidyl label ordetection tag, or a clearance-modifying agent such as various isomers ofpolyethylene glycol, a peptide that binds to a third component, oranother carbohydrate or lipophilic agent.

Uses of Cysteine Engineered Anti-TENB2 Antibodies

Cysteine engineered anti-TENB2 antibodies, and conjugates thereof mayfind use as therapeutic and/or diagnostic agents. The present inventionfurther provides methods of preventing, managing, treating orameliorating one or more symptoms associated with a TENB2 relateddisorder. In particular, the present invention provides methods ofpreventing, managing, treating, or ameliorating one or more symptomsassociated with a cell proliferative disorder, such as cancer, e.g.,ovarian cancer, cervical cancer, uterine cancer, pancreatic cancer, lungcancer and breast cancer. The present invention still further providesmethods for diagnosing a TENB2 related disorder or predisposition todeveloping such a disorder, as well as methods for identifyingantibodies, and antigen-binding fragments of antibodies, thatpreferentially bind cell-associated TENB2 polypeptides.

Another embodiment of the present invention is directed to the use of acysteine engineered anti-TENB2 antibody for the preparation of amedicament useful in the treatment of a condition which is responsive toa TENB2 related disorder.

Cysteine Engineered Anti-TENB2 Antibody Drug Conjugates

Another aspect of the invention is an antibody-drug conjugate compoundcomprising a cysteine engineered anti-TENB2 antibody (Ab), and anauristatin drug moiety (D) wherein the cysteine engineered antibody isattached through one or more free cysteine amino acids by a linkermoiety (L) to D; the compound having Formula I:

Ab-(L-D)_(p)  I

where p is 1, 2, 3, or 4; and wherein the cysteine engineered antibodyis prepared by a process comprising replacing one or more amino acidresidues of a parent anti-TENB2 antibody by one or more free cysteineamino acids.

FIG. 5 shows embodiments of cysteine engineered anti-TENB2 antibody drugconjugates (ADC) where an auristatin drug moiety is attached to anengineered cysteine group in: the light chain (LC-ADC); the heavy chain(HC-ADC); and the Fc region (Fc-ADC).

Potential advantages of cysteine engineered anti-TENB2 antibody drugconjugates include improved safety (larger therapeutic index), improvedPK parameters, the antibody interchain disulfide bonds are retainedwhich may stabilize the conjugate and retain its active bindingconformation, the sites of drug conjugation are defined, and thepreparation of cysteine engineered antibody drug conjugates fromconjugation of cysteine engineered antibodies to drug-linker reagentsresults in a more homogeneous product.

Drug Moieties

Auristatin drug moieties of the antibody-drug conjugates (ADC) ofFormula I include dolastatins, auristatins (U.S. Pat. No. 5,635,483;U.S. Pat. No. 5,780,588; U.S. Pat. No. 5,767,237; U.S. Pat. No.6,124,431), and analogs and derivatives thereof. Dolastatins andauristatins have been shown to interfere with microtubule dynamics, GTPhydrolysis, and nuclear and cellular division (Woyke et al (2001)Antimicrob. Agents and Chemother. 45(12):3580-3584) and have anticancer(U.S. Pat. No. 5,663,149) and antifungal activity (Pettit et al (1998)Antimicrob. Agents Chemother. 42:2961-2965). Various forms of adolastatin or auristatin drug moiety may be covalently attached to anantibody through the N (amino) terminus or the C (carboxyl) terminus ofthe peptidic drug moiety (WO 02/088172; Doronina et al (2003) NatureBiotechnology 21(7):778-784; Francisco et al (2003) Blood 102(4):1458-1465).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in: WO2005/081711; Senter et al, Proceedings of the American Association forCancer Research, Volume 45, Abstract Number 623, presented Mar. 28,2004, the disclosure of each which are expressly incorporated byreference in their entirety. Exemplary auristatin drug moieties includeMMAE, and MMAF.

The auristatin drug moiety (D) of the antibody-drug conjugates (ADC) ofFormula I include the monomethylauristatin drug moieties MMAE and MMAF.The N-terminus of the MMAE or MMAF drug moiety is covalently attachedvia a linker to a engineered cysteine of the antibody.

Other exemplary auristatin drug moieties include monomethylvalinecompounds having phenylalanine carboxy modifications at the C-terminusof the pentapeptide auristatin drug moiety (WO 2007/008848) andmonomethylvaline compounds having phenylalanine sidechain modificationsat the C-terminus of the pentapeptide auristatin drug moiety (WO2007/008603).

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schröder and K. Lübke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry.

Linkers

“Linker”, “Linker Unit”, or “link” means a chemical moiety comprising acovalent bond or a chain of atoms that covalently attaches an antibodyto a drug moiety. In various embodiments, a linker is specified as L. A“Linker” (L) is a bifunctional or multifunctional moiety which can beused to link one or more Drug moieties (D) and an antibody unit (Ab) toform antibody-drug conjugates (ADC) of Formula I. Antibody-drugconjugates (ADC) can be conveniently prepared using a Linker havingreactive functionality for binding to the Drug and to the Antibody. Acysteine thiol of a cysteine engineered antibody (Ab) can form a bondwith an electrophilic functional group of a linker reagent, a drugmoiety or drug-linker intermediate.

In one aspect, a Linker has a reactive site which has an electrophilicgroup that is reactive to a nucleophilic cysteine present on anantibody. The cysteine thiol of the antibody is reactive with anelectrophilic group on a Linker and forms a covalent bond to a Linker.Useful electrophilic groups include, but are not limited to, maleimideand haloacetamide groups.

Linkers include a divalent radical such as an alkyldiyl, an arylene, aheteroarylene, moieties such as: —(CR₂)_(n)O(CR₂)_(n)—, repeating unitsof alkyloxy (e.g. polyethylenoxy, PEG, polymethyleneoxy) and alkylamino(e.g. polyethyleneamino, Jeffamine™); and diacid ester and amidesincluding succinate, succinamide, diglycolate, malonate, and caproamide.

Cysteine engineered antibodies react with linker reagents or drug-linkerintermediates, with electrophilic functional groups such as maleimide orα-halo carbonyl, according to the conjugation method at page 766 ofKlussman, et al (2004), Bioconjugate Chemistry 15(4):765-773, andaccording to the protocol of Example 3.

The linker may be composed of one or more linker components. Exemplarylinker components include 6-maleimidocaproyl (“MC”), maleimidopropanoyl(“MP”), valine-citrulline (“val-cit” or “vc”), alanine-phenylalanine(“ala-phe” or “af”), p-aminobenzyloxycarbonyl (“PAB”), N-succinimidyl4-(2-pyridylthio) pentanoate (“SPP”), N-succinimidyl4-(N-maleimidomethyl) cyclohexane-1 carboxylate (“SMCC’), N-Succinimidyl(4-iodo-acetyl) aminobenzoate (“SIAB”), ethyleneoxy —CH₂CH₂O— as one ormore repeating units (“EO” or “PEO”). Additional linker components areknown in the art and some are described herein.

In one embodiment, linker L of an ADC has the formula:

-A_(a)-W_(w)—Y_(y)—

wherein:

-A- is a Stretcher unit covalently attached to a cysteine thiol of theantibody (Ab);

a is 0 or 1;

each —W— is independently an Amino Acid unit;

w is independently an integer ranging from 0 to 12;

—Y— is a Spacer unit covalently attached to the drug moiety; and

-   -   y is 0, 1 or 2.

Stretcher Unit

The Stretcher unit (-A-), when present, is capable of linking anantibody unit to an amino acid unit (—W—). In this regard an antibody(Ab) has a free cysteine thiol group that can form a bond with anelectrophilic functional group of a Stretcher Unit. Exemplary stretcherunits in Formula I conjugates are depicted by Formulas II and III,wherein Ab-, —W—, —Y—, -D, w and y are as defined above, and R¹⁷ is adivalent radical selected from (CH₂)_(r), C₃-C₈ carbocyclyl,O—(CH₂)_(r), arylene, (CH₂)_(r)-arylene, -arylene-(CH₂)_(r)—,(CH₂)_(r)—(C₃-C₈ carbocyclyl), (C₃-C₈ carbocyclyl)-(CH₂)_(r)—, C₃-C₈heterocyclyl, (CH₂)_(r)—(C₃-C₈ heterocyclyl), —(C₃-C₈heterocyclyl)-(CH₂)_(r)—, —(CH₂)_(r)C(O)NR^(b)(CH₂)_(r)—,—(CH₂CH₂O)_(r)—, —(CH₂CH₂O)_(r)—CH₂—,—(CH₂)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—,—(CH₂)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—CH₂—,—(CH₂CH₂O)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—,—(CH₂CH₂O)_(r)C(O)NR^(b)(CH₂H₂O)_(r)—CH₂—, and—(CH₂CH₂O)_(r)C(O)NR^(b)(CH₂)_(r)—; where R^(b) is H, C₁-C₆ alkyl,phenyl, or benzyl; and r is independently an integer ranging from 1-10.

Arylene includes divalent aromatic hydrocarbon radicals of 6-20 carbonatoms derived by the removal of two hydrogen atoms from the aromaticring system. Typical arylene groups include, but are not limited to,radicals derived from benzene, substituted benzene, naphthalene,anthracene, biphenyl, and the like.

Heterocyclyl groups include a ring system in which one or more ringatoms is a heteroatom, e.g. nitrogen, oxygen, and sulfur. Theheterocycle radical comprises 1 to 20 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S. A heterocycle may be amonocycle having 3 to 7 ring members (2 to 6 carbon atoms and 1 to 3heteroatoms selected from N, O, P, and S) or a bicycle having 7 to 10ring members (4 to 9 carbon atoms and 1 to 3 heteroatoms selected fromN, O, P, and S), for example: a bicyclo[4,5], [5,5], [5,6], or system.Heterocycles are described in Paquette, Leo A.; “Principles of ModernHeterocyclic Chemistry” (W. A. Benjamin, New York, 1968), particularlyChapters 1, 3, 4, 6, 7, and 9; “The Chemistry of Heterocyclic Compounds,A series of Monographs” (John Wiley & Sons, New York, 1950 to present),in particular Volumes 13, 14, 16, 19, and 28; and J. Am. Chem. Soc.(1960) 82:5566.

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, bis-tetrahydrofuranyl,tetrahydropyranyl, bis-tetrahydropyranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazolyl, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4Ah-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,and isatinoyl.

Carbocyclyl groups include a saturated or unsaturated ring having 3 to 7carbon atoms as a monocycle or 7 to 12 carbon atoms as a bicycle.Monocyclic carbocycles have 3 to 6 ring atoms, still more typically 5 or6 ring atoms. Bicyclic carbocycles have 7 to 12 ring atoms, e.g.arranged as a bicyclo[4,5], [5,5], [5,6] or [6,6] system, or 9 or 10ring atoms arranged as a bicyclo[5,6] or [6,6] system. Examples ofmonocyclic carbocycles include cyclopropyl, cyclobutyl, cyclopentyl,1-cyclopent-1-enyl, 1-cyclopent-2-enyl, 1-cyclopent-3-enyl, cyclohexyl,1-cyclohex-1-enyl, 1-cyclohex-2-enyl, 1-cyclohex-3-enyl, cycloheptyl,and cyclooctyl.

It is to be understood from all the exemplary embodiments of Formula IADC such as II-V, that even where not denoted expressly, from 1 to 4drug moieties are linked to an antibody (p=1-4), depending on the numberof engineered cysteine residues.

An illustrative Formula II Stretcher unit is derived frommaleimidocaproyl (MC) wherein R¹⁷ is —(CH₂)₅—:

An illustrative Stretcher unit of Formula II, and is derived frommaleimido-propanoyl (MP) wherein R¹⁷ is —(CH₂)₂—:

Another illustrative Stretcher unit of Formula II wherein R¹⁷ is—(CH₂CH₂O)_(r)—CH₂— and r is 2:

Another illustrative Stretcher unit of Formula II wherein R⁷ is—(CH₂)_(r)C(O)NR^(b)(CH₂CH₂O)_(r)—CH₂— where R^(b) is H and each r is 2:

An illustrative Stretcher unit of Formula III wherein R¹⁷ is —(CH₂)₅—:

In another embodiment, the Stretcher unit is linked to the cysteineengineered anti-TENB2 antibody via a disulfide bond between theengineered cysteine sulfur atom of the antibody and a sulfur atom of theStretcher unit. A representative Stretcher unit of this embodiment isdepicted by Formula IV, wherein R¹⁷, Ab-, —W—, —Y—, -D, w and y are asdefined above.

Ab-SS—R¹⁷—C(O)—W_(w)—Y_(y)-D)_(p)  IV

In yet another embodiment, the reactive group of the Stretcher containsa thiol-reactive functional group that can form a bond with a freecysteine thiol of an antibody. Examples of thiol-reaction functionalgroups include, but are not limited to, maleimide, α-haloacetyl,activated esters such as succinimide esters, 4-nitrophenyl esters,pentafluorophenyl esters, tetrafluorophenyl esters, anhydrides, acidchlorides, sulfonyl chlorides, isocyanates and isothiocyanates.Representative Stretcher units of this embodiment are depicted byFormulas Va and Vb, wherein —R¹⁷—, Ab-, —W—, —Y—, -D, w and y are asdefined above;

Ab-SC(O)NH—R¹⁷—C(O)—W_(w)—Y_(y)-D)_(p)  Va

Ab-SC(S)NH—R¹⁷—C(O)—W_(w)—Y_(y)-D)_(p)  Vb

In another embodiment, the linker may be a dendritic type linker forcovalent attachment of more than one drug moiety through a branching,multifunctional linker moiety to an antibody (Sun et al (2002)Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003)Bioorganic & Medicinal Chemistry 11:1761-1768; King (2002) TetrahedronLetters 43:1987-1990). Dendritic linkers can increase the molar ratio ofdrug to antibody, i.e. loading, which is related to the potency of theADC. Thus, where a cysteine engineered antibody bears only one reactivecysteine thiol group, a multitude of drug moieties may be attachedthrough a dendritic linker.

Amino Acid Unit

The linker may comprise amino acid residues. The Amino Acid unit(—W_(w)-), when present, links the antibody (Ab) to the drug moiety (D)of the cysteine engineered antibody-drug conjugate (ADC) of theinvention.

—W_(w)— is a dipeptide, tripeptide, tetrapeptide, pentapeptide,hexapeptide, heptapeptide, octapeptide, nonapeptide, decapeptide,undecapeptide or dodecapeptide unit. Amino acid residues which comprisethe Amino Acid unit include those occurring naturally, as well as minoramino acids and non-naturally occurring amino acid analogs, such ascitrulline. Each —W— unit independently has the formula denoted below inthe square brackets, and w is an integer ranging from 0 to 12:

wherein R¹⁹ is hydrogen, methyl, isopropyl, isobutyl, sec-butyl, benzyl,p-hydroxybenzyl, —CH₂OH, —CH(OH)CH₃, —CH₂CH₂SCH₃, —CH₂CONH₂, —CH₂COOH,—CH₂CH₂CONH₂, —CH₂CH₂COOH, —(CH₂)₃NHC(═NH)NH₂, —(CH₂)₃NH₂,—(CH₂)₃NHCOCH₃, —(CH₂)₃NHCHO, —(CH₂)₄NHC(═NH)NH₂, —(CH₂)₄NH2,—(CH₂)₄NHCOCH₃, —(CH₂)₄NHCHO, —(CH₂)₃NHCONH₂, —(CH₂)₄NHCONH₂,—CH₂CH₂CH(OH)CH₂NH₂, 2-pyridylmethyl-, 3-pyridylmethyl-,4-pyridylmethyl-, phenyl, cyclohexyl,

When R¹⁹ is other than hydrogen, the carbon atom to which R¹⁹ isattached is chiral. Each carbon atom to which R¹⁹ is attached isindependently in the (S) or (R) configuration, or a racemic mixture.Amino acid units may thus be enantiomerically pure, racemic, ordiastereomeric.

Exemplary —W_(w)— Amino Acid units include a dipeptide, a tripeptide, atetrapeptide or a pentapeptide. Exemplary dipeptides include:valine-citrulline (vc or val-cit), alanine-phenylalanine (af orala-phe). Exemplary tripeptides include: glycine-valine-citrulline(gly-val-cit) and glycine-glycine-glycine (gly-gly-gly). Amino acidresidues which comprise an amino acid linker component include thoseoccurring naturally, as well as minor amino acids and non-naturallyoccurring amino acid analogs, such as citrulline.

The Amino Acid unit can be enzymatically cleaved by one or more enzymes,including a tumor-associated protease, to liberate the Drug moiety (-D),which in one embodiment is protonated in vivo upon release to provide aDrug (D). Amino acid linker components can be designed and optimized intheir selectivity for enzymatic cleavage by a particular enzymes, forexample, a tumor-associated protease, cathepsin B, C and D, or a plasminprotease.

Spacer Unit

The Spacer unit (—Y_(y)—), when present (y=1 or 2), links an Amino Acidunit (—W_(w)—) to the drug moiety (D) when an Amino Acid unit is present(w=1-12). Alternately, the Spacer unit links the Stretcher unit to theDrug moiety when the Amino Acid unit is absent. The Spacer unit alsolinks the drug moiety to the antibody unit when both the Amino Acid unitand Stretcher unit are absent (w, y=0). Spacer units are of two generaltypes: self-immolative and non self-immolative. A non self-immolativeSpacer unit is one in which part or all of the Spacer unit remains boundto the Drug moiety after cleavage, particularly enzymatic, of an AminoAcid unit from the antibody-drug conjugate or the Drug moiety-linker.When an ADC containing a glycine-glycine Spacer unit or a glycine Spacerunit undergoes enzymatic cleavage via a tumor-cell associated-protease,a cancer-cell-associated protease or a lymphocyte-associated protease, aglycine-glycine-Drug moiety or a glycine-Drug moiety is cleaved fromAb-A_(a)-Ww-. In one embodiment, an independent hydrolysis reactiontakes place within the target cell, cleaving the glycine-Drug moietybond and liberating the Drug.

In another embodiment, —Y_(y)— is a p-aminobenzylcarbamoyl (PAB) unitwhose phenylene portion is substituted with Q_(m) wherein Q is —C₁-C₈alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano; and m is an integerranging from 0-4.

Exemplary embodiments of a non self-immolative Spacer unit (—Y—) are:-Gly-Gly-; -Gly-; -Ala-Phe-; -Val-Cit-.

In one embodiment, a Drug moiety-linker or an ADC is provided in whichthe Spacer unit is absent (y=−0), or a pharmaceutically acceptable saltor solvate thereof.

Alternatively, an ADC containing a self-immolative Spacer unit canrelease -D. In one embodiment, —Y— is a PAB group that is linked to—W_(w)— via the amino nitrogen atom of the PAB group, and connecteddirectly to -D via a carbonate, carbamate or ether group, where the ADChas the exemplary structure:

wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;m is an integer ranging from 0-4; and p ranges from 1 to 4.

Other examples of self-immolative spacers include, but are not limitedto, aromatic compounds that are electronically similar to the PAB groupsuch as 2-aminoimidazol-5-methanol derivatives (Hay et al. (1999)Bioorg. Med. Chem. Lett. 9:2237), heterocyclic PAB analogs (US2005/0256030), beta-glucuronide (WO 2007/011968), and ortho orpara-aminobenzylacetals. Spacers can be used that undergo cyclizationupon amide bond hydrolysis, such as substituted and unsubstituted4-aminobutyric acid amides (Rodrigues et al (1995) Chemistry Biology2:223), appropriately substituted bicyclo[2.2.1] and bicyclo[2.2.2] ringsystems (Storm et al (1972) J. Amer. Chem. Soc. 94:5815) and2-aminophenylpropionic acid amides (Amsberry, et al (1990) J. Org. Chem.55:5867). Elimination of amine-containing drugs that are substituted atglycine (Kingsbury et al (1984) J. Med. Chem. 27:1447) are also examplesof self-immolative spacer useful in ADCs.

Exemplary Spacer units (—Y_(y)—) are represented by Formulas X-XII:

In another embodiment, linker L may be a dendritic type linker forcovalent attachment of more than one drug moiety through a branching,multifunctional linker moiety to an antibody (Sun et al (2002)Bioorganic & Medicinal Chemistry Letters 12:2213-2215; Sun et al (2003)Bioorganic & Medicinal Chemistry 11:1761-1768). Dendritic linkers canincrease the molar ratio of drug to antibody, i.e. loading, which isrelated to the potency of the ADC. Thus, where a cysteine engineeredantibody bears only one reactive cysteine thiol group, a multitude ofdrug moieties may be attached through a dendritic linker. Exemplaryembodiments of branched, dendritic linkers include2,6-bis(hydroxymethyl)-p-cresol and 2,4,6-tris(hydroxymethyl)-phenoldendrimer units (WO 2004/01993; Szalai et al (2003) J. Amer. Chem. Soc.125:15688-15689; Shamis et al (2004) J. Amer. Chem. Soc. 126:1726-1731;Amir et al (2003) Angew. Chem. Int. Ed. 42:4494-4499).

In one embodiment, the Spacer unit is a branchedbis(hydroxymethyl)styrene (BHMS), which can be used to incorporate andrelease multiple drugs, having the structure:

comprising a 2-(4-aminobenzylidene)propane-1,3-diol dendrimer unit (WO2004/043493; de Groot et al (2003) Angew. Chem. Int. Ed. 42:4490-4494),wherein Q is —C₁-C₈ alkyl, —O—(C₁-C₈ alkyl), -halogen, -nitro or -cyano;m is an integer ranging from 0-4; n is 0 or 1; and p ranges ranging from1 to 4.

Exemplary embodiments of the Formula I antibody-drug conjugate compoundsinclude XIIIa (MC), XIIIb (val-cit), XIIIc (MC-val-cit), and XIIId(MC-val-cit-PAB):

Other exemplary embodiments of the Formula Ia antibody-drug conjugatecompounds include XIVa-e:

Y is:

where R is independently H or C₁-C₆ alkyl; and n is 1 to 12.

In another embodiment, a Linker has a reactive functional group whichhas a nucleophilic group that is reactive to an electrophilic grouppresent on an antibody. Useful electrophilic groups on an antibodyinclude, but are not limited to, aldehyde and ketone carbonyl groups.The heteroatom of a nucleophilic group of a Linker can react with anelectrophilic group on an antibody and form a covalent bond to anantibody unit. Useful nucleophilic groups on a Linker include, but arenot limited to, hydrazide, oxime, amino, hydrazine, thiosemicarbazone,hydrazine carboxylate, and arylhydrazide. The electrophilic group on anantibody provides a convenient site for attachment to a Linker.

Typically, peptide-type Linkers can be prepared by forming a peptidebond between two or more amino acids and/or peptide fragments. Suchpeptide bonds can be prepared, for example, according to the liquidphase synthesis method (E. Schröder and K. Lübke (1965) “The Peptides”,volume 1, pp 76-136, Academic Press) which is well known in the field ofpeptide chemistry. Linker intermediates may be assembled with anycombination or sequence of reactions including Spacer, Stretcher, andAmino Acid units. The Spacer, Stretcher, and Amino Acid units may employreactive functional groups which are electrophilic, nucleophilic, orfree radical in nature. Reactive functional groups include, but are notlimited to carboxyls, hydroxyls, para-nitrophenylcarbonate,isothiocyanate, and leaving groups, such as O-mesyl, O-tosyl, —Cl, —Br,—I; or maleimide.

In another embodiment, the Linker may be substituted with groups whichmodulated solubility or reactivity. For example, a charged substituentsuch as sulfonate (—SO₃ ⁻) or ammonium, may increase water solubility ofthe reagent and facilitate the coupling reaction of the linker reagentwith the antibody or the drug moiety, or facilitate the couplingreaction of Ab-L (antibody-linker intermediate) with D, or D-L(drug-linker intermediate) with Ab, depending on the synthetic routeemployed to prepare the ADC.

Linker Reagents

Conjugates of the antibody and auristatin may be made using a variety ofbifunctional linker reagents such as N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate (SMCC), iminothiolane (IT), bifunctionalderivatives of imidoesters (such as dimethyl adipimidate HCl), activeesters (such as disuccinimidyl suberate), aldehydes (such asglutaraldehyde), bis-azido compounds (such as bis (p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astoluene 2,6-diisocyanate), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene).

The antibody drug conjugates may also be prepared with linker reagents:BMPEO, BMPS, EMCS, GMBS, HBVS, LC-SMCC, MBS, MPBH, SBAP, SIA, SIAB,SMPB, SMPH, sulfo-EMCS, sulfo-GMBS, sulfo-KMUS, sulfo-MBS, sulfo-SIAB,sulfo-SMCC, and sulfo-SMPB, and SVSB(succinimidyl-(4-vinylsulfone)benzoate), and including bis-maleimidereagents: DTME, BMB, BMDB, BMH, BMOE, BM(PEO)₃, and BM(PEO)₄, which arecommercially available from Pierce Biotechnology, Inc., P.O. Box 117,Rockford, Ill. 61105, USA. Bis-maleimide reagents allow the attachmentof the thiol group of a cysteine engineered antibody to athiol-containing drug moiety, label, or linker intermediate, in asequential or concurrent fashion. Other functional groups besidesmaleimide, which are reactive with a thiol group of a cysteineengineered antibody, drug moiety, label, or linker intermediate includeiodoacetamide, bromoacetamide, vinyl pyridine, disulfide, pyridyldisulfide, isocyanate, and isothiocyanate.

Useful linker reagents can also be obtained via other commercialsources, such as Molecular Biosciences Inc. (Boulder, Colo.), orsynthesized in accordance with procedures described in Toki et al (2002)J. Org. Chem. 67:1866-1872; Walker, M. A. (1995) J. Org. Chem.60:5352-5355; Frisch et al (1996) Bioconjugate Chem. 7:180-186; U.S.Pat. No. 6,214,345; WO 02/088172; US 2003130189; US2003096743; WO03/026577; WO 03/043583; and WO 04/032828.

Stretchers of formula (IIIa) can be introduced into a Linker by reactingthe following linker reagents with the N-terminus of an Amino Acid unit:

where n is an integer ranging from 1-10 and T is —H or —SO₃Na;

where n is an integer ranging from 0-3;

Stretcher units of can be introduced into a Linker by reacting thefollowing bifunctional reagents with the N-terminus of an Amino Acidunit:

where X is Br or I.

Stretcher units of formula can also be introduced into a Linker byreacting the following bifunctional reagents with the N-terminus of anAmino Acid unit:

An exemplary valine-citrulline (val-cit or vc) dipeptide linker reagenthaving a maleimide Stretcher and a para-aminobenzylcarbamoyl (PAB)self-immolative Spacer has the structure:

An exemplary phe-lys(Mtr, mono-4-methoxytrityl) dipeptide linker reagenthaving a maleimide Stretcher unit and a PAB self-immolative Spacer unitcan be prepared according to Dubowchik, et al. (1997) TetrahedronLetters, 38:5257-60, and has the structure:

Exemplary drug-linker intermediates include:

Exemplary antibody-drug conjugate compounds of the invention include:

where Val is valine; Cit is citrulline; p is 1, 2, 3, or 4; and Ab is acysteine engineered anti-TENB2 antibody.

Preparation of Cysteine Engineered Anti-TENB2 Antibody-Drug Conjugates

The ADC of Formula I may be prepared by several routes, employingorganic chemistry reactions, conditions, and reagents known to thoseskilled in the art, including: (1) reaction of a cysteine group of acysteine engineered antibody with a linker reagent, to formantibody-linker intermediate Ab-L, via a covalent bond, followed byreaction with an activated drug moiety D; and (2) reaction of anucleophilic group of a drug moiety with a linker reagent, to formdrug-linker intermediate D-L, via a covalent bond, followed by reactionwith a cysteine group of a cysteine engineered antibody. Conjugationmethods (1) and (2) may be employed with a variety of cysteineengineered antibodies, drug moieties, and linkers to prepare theantibody-drug conjugates of Formula I.

Antibody cysteine thiol groups are nucleophilic and capable of reactingto form covalent bonds with electrophilic groups on linker reagents anddrug-linker intermediates including: (i) active esters such as NHSesters, HOBt esters, haloformates, and acid halides; (ii) alkyl andbenzyl halides, such as haloacetamides; (iii) aldehydes, ketones,carboxyl, and maleimide groups; and (iv) disulfides, including pyridyldisulfides, via sulfide exchange. Nucleophilic groups on a drug moietyinclude, but are not limited to: amine, thiol, hydroxyl, hydrazide,oxime, hydrazine, thiosemicarbazone, hydrazine carboxylate, andarylhydrazide groups capable of reacting to form covalent bonds withelectrophilic groups on linker moieties and linker reagents.

Cysteine engineered antibodies may be made reactive for conjugation withlinker reagents by treatment with a reducing agent such as DTT(Cleland's reagent, dithiothreitol) or TCEP(tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999) Anal.Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.), followed byreoxidation to reform interchain and intrachain disulfide bonds (Example2). For example, full length, cysteine engineered monoclonal antibodies(ThioMabs) expressed in CHO cells are reduced with about a 50 foldexcess of TCEP for 3 hrs at 37° C. to reduce disulfide bonds in cysteineadducts which may form between the newly introduced cysteine residuesand the cysteine present in the culture media. The reduced ThioMab isdiluted and loaded onto HiTrap S column in 10 mM sodium acetate, pH 5,and eluted with PBS containing 0.3M sodium chloride. Disulfide bondswere reestablished between cysteine residues present in the parent Mabwith dilute (200 nM) aqueous copper sulfate (CuSO₄) at room temperature,overnight. Alternatively, dehydroascorbic acid (DHAA) is an effectiveoxidant to reestablish the intrachain disulfide groups of the cysteineengineered antibody after reductive cleavage of the cysteine adducts.Other oxidants, i.e. oxidizing agents, and oxidizing conditions, whichare known in the art may be used. Ambient air oxidation is alsoeffective. This mild, partial reoxidation step forms intrachaindisulfides efficiently with high fidelity and preserves the thiol groupsof the newly introduced cysteine residues. An approximate 3 fold excessof drug-linker intermediate, e.g. MC-vc-PAB-MMAE, relative to antibody(about 1.5 fold excess relative to newly introduced cysteine residues)was added, mixed, and let stand for about an hour at room temperature toeffect conjugation and form the TMEFF2#19 anti-TENB2 antibody-drugconjugate. The conjugation mixture was gel filtered and loaded andeluted through a HiTrap S column to remove excess drug-linkerintermediate and other impurities.

FIG. 6 shows the general process to prepare a cysteine engineeredantibody expressed from cell culture for conjugation. When the cellculture media contains cysteine, disulfide adducts can form between thenewly introduced cysteine amino acid and cysteine from media. Thesecysteine adducts, depicted as a circle in the exemplary ThioMab (left)in FIG. 6, must be reduced to generate cysteine engineered antibodiesreactive for conjugation. Cysteine adducts, presumably along withvarious interchain disulfide bonds, are reductively cleaved to give areduced form of the antibody with reducing agents such as TCEP. Theinterchain disulfide bonds between paired cysteine residues are reformedunder partial oxidation conditions with copper sulfate, DHAA, orexposure to ambient oxygen. The newly introduced, engineered, andunpaired cysteine residues remain available for reaction with linkerreagents or drug-linker intermediates to form the antibody conjugates ofthe invention. The ThioMabs expressed in mammalian cell lines result inexternally conjugated Cys adduct to an engineered Cys through —S—S— bondformation. Hence the purified ThioMabs are treated with the reductionand reoxidation procedures as described in Example 2 to produce reactiveThioMabs. These ThioMabs are used to conjugate with maleimide containingcytotoxic drugs, fluorophores, and other labels.

Analysis of cysteine engineered antibody drug conjugate reactions showdecreased heterogeneity relative to antibody drug conjugates prepared byreduction of interchain or intrachain disulfide bonds followed byconjugation (standard ADC) with a thiol reactive drug linkerintermediate.

Methods of Screening

Yet another embodiment of the present invention is directed to a methodof determining the presence of a TENB2 polypeptide in a sample suspectedof containing the TENB2 polypeptide, wherein the method comprisesexposing the sample to a cysteine engineered anti-TENB2 antibody, orantibody drug conjugate thereof, that binds to the TENB2 polypeptide anddetermining binding of the cysteine engineered anti-TENB2 antibody, orantibody drug conjugate thereof, to the TENB2 polypeptide in the sample,wherein the presence of such binding is indicative of the presence ofthe TENB2 polypeptide in the sample. Optionally, the sample may containcells (which may be cancer cells) suspected of expressing the TENB2polypeptide. The cysteine engineered anti-TENB2 antibody, or antibodydrug conjugate thereof, employed in the method may optionally bedetectably labeled, attached to a solid support, or the like.

Another embodiment of the present invention is directed to a method ofdiagnosing the presence of a tumor in a mammal, wherein the methodcomprises (a) contacting a test sample comprising tissue cells obtainedfrom the mammal with a cysteine engineered anti-TENB2 antibody, orantibody drug conjugate thereof, that binds to a TENB2 polypeptide and(b) detecting the formation of a complex between the cysteine engineeredanti-TENB2 antibody, or antibody drug conjugate thereof, and the TENB2polypeptide in the test sample, wherein the formation of a complex isindicative of the presence of a tumor in the mammal. Optionally, thecysteine engineered anti-TENB2 antibody, or antibody drug conjugatethereof, is detectably labeled, attached to a solid support, or thelike, and/or the test sample of tissue cells is obtained from anindividual suspected of having a cancerous tumor.

In Vitro Cell Proliferation Assays

One embodiment of the present invention is directed to a method forinhibiting the growth of a cell that expresses a TENB2 polypeptide,wherein the method comprises contacting the cell with a cysteineengineered anti-TENB2 antibody, or antibody drug conjugate thereof, tothe TENB2 polypeptide causes inhibition of the growth of the cellexpressing the TENB2. The cell may be a cancer cell and binding of thecysteine engineered antibody, or antibody drug conjugate thereof, to theTENB2 polypeptide causes death of the cell expressing the TENB2polypeptide.

Generally, the cytotoxic or cytostatic activity of an antibody-drugconjugate (ADC) is measured by: exposing mammalian cells expressingTENB2 polypeptide to ADC in a cell culture medium; culturing the cellsfor a period from about 6 hours to about 5 days; and measuring cellviability. Mammalian cells useful for cell proliferation assays include:(1) a TENB2 polypeptide-expressing LuCaP77 tumor xenograft; (2) aPC3-derived cell line engineered to stably express a portion of theTENB2 polypeptide on its cell surface (PC3/TENB2); and (3) a PC3 cellline that does not express TENB2 polypeptide (PC3/neo). Cell-based invitro assays are used to measure viability (proliferation),cytotoxicity, and induction of apoptosis (caspase activation) of the ADCof the invention.

Pharmacokinetics—Serum Clearance and Stability

The disposition of the anti-TENB2 antibody-drug conjugates in vivo wasanalyzed by measuring the serum concentrations of antibody and of drugconjugate after a single intravenous bolus dose into Sprague-Dawleyrats. Concentrations of antibody-drug conjugates bearing at least onecytotoxic drug were measured with an ELISA that used anti-MMAE for thecapture and biotinylated TENB2 extra-cellular domain (ECD) andstreptavidin-horseradish peroxidase (HRP) for detection. Total TMEFF2#19and ThioTMEFF2#19 concentrations in serum were measured with an ELISAthat used TENB2 ECD for capture and anti-□human-Fc HRP as the secondaryantibody. This assay measured any anti-TENB2 antibody, both with andwithout conjugated MMAE. The assays have lower limits of quantitation of16.4 ng/mL with a minimum dilution of 1:100. The serumconcentration-time data from each animal was analyzed using atwo-compartment model with IV bolus input, first-order elimination, andmacro-rate constants (Model 8, WinNonlin Pro v.5.0.1, PharsightCorporation, Mountain View, Calif.). Overall goodness of fit was basedon the predicted estimate, standard error for the prediction, andpercentage of coefficient of variation for primary and secondaryparameters, as well as inspection of residual plots between observed andpredicted concentration □-time data. Individual primary PK parameterscomprised the zero-time intercepts (A and B) associated with the alphaand beta phases, respectively, and the micro-rate constants (alpha andbeta). The following modeling options were used: Initial estimates weredetermined using WinNonlin; Concentrations were weighted by thereciprocal of the predicted concentration squared (1/ŷ²); Nelder-Meadminimization algorithm was used. The following PK parameters werereported: AUC_(0□INF), CL, C_(max), MRT, t_(1/2,a), t_(1/2,b), V₁ andV_(ss).

Results of 28-day pharmacokinetics analyses in rats are shown in FIG.15. Rats were dosed with 5 mg/kg body weight of thio TMEFF2#19-VC-MMAEor 5 mg/kg TMEFF2#19-VC-MMAE. Serum from rats was collected at 5minutes, 1 hour, 6 hours, 24 hours, and 2, 3, 4, 8, 11, 15, 21, and 28days after dosing. Dose linearity of kinetics was observed for chTMEFF2#19-VC-MMAE between 0.5 and 5 mg/kg dose, so the 5 mg/kg dose datahave been arithmetically converted to reflect the predicted data at 5mg/kg for comparison with thio TMEFF2#19-VC-MMAE.

Rodent Toxicity

The toxicity of cysteine engineered anti-TENB2 antibody-drug conjugateswas evaluated in an acute toxicity rat and cynomolgus models. Toxicityof ADC was investigated by treatment of female Sprague-Dawley rats andcynomolgus monkeys with the ADC and subsequent inspection and analysisof the effects on various organs. Based on gross observations (bodyweights), clinical pathology parameters (serum chemistry and hematology)and histopathology, the toxicity of ADC may be observed, characterized,and measured. It was found that at equivalent dose levels, notarget-dependant effects appeared. Target-independent toxicities wereobserved at doe that exceeded the efficacious doses in animal tumormodels.

Methods of Treatment

Another embodiment of the present invention is directed to a method oftherapeutically treating a mammal having a cancerous tumor comprisingcells that express a TENB2 polypeptide, wherein the method comprisesadministering to the mammal a therapeutically effective amount of acysteine engineered antibody, or antibody drug conjugate thereof, thatbinds to the TENB2 polypeptide, thereby resulting in the effectivetherapeutic treatment of the tumor.

Another embodiment of the present invention is directed to a method fortreating or preventing a cell proliferative disorder associated withaltered, preferably increased, expression or activity of a TENB2polypeptide, the method comprising administering to a subject in need ofsuch treatment an effective amount of a cysteine engineered anti-TENB2antibody, or antibody drug conjugate thereof. An exemplary cellproliferative disorder is cancer. Effective treatment or prevention ofthe cell proliferative disorder may be a result of direct killing orgrowth inhibition of cells that express a TENB2 polypeptide or byantagonizing the cell growth potentiating activity of a TENB2polypeptide with the cysteine engineered anti-TENB2 antibody, orantibody drug conjugate thereof.

Yet another embodiment of the present invention is directed to a methodof binding a cysteine engineered anti-TENB2 antibody, or antibody drugconjugate thereof, to a cell that expresses a TENB2 polypeptide, whereinthe method comprises contacting a cell that expresses a TENB2polypeptide with said cysteine engineered anti-TENB2 antibody, orantibody drug conjugate thereof, under conditions which are suitable forbinding of the cysteine engineered anti-TENB2 antibody, or antibody drugconjugate thereof, to said TENB2 polypeptide and allowing bindingtherebetween. In preferred embodiments, the cysteine engineeredanti-TENB2 antibody, or antibody drug conjugate thereof, is labeled witha molecule or compound that is useful for qualitatively and/orquantitatively determining the location and/or amount of binding of thecysteine engineered anti-TENB2 antibody, or antibody drug conjugatethereof, to the cell.

Other embodiments of the present invention are directed to the use of acysteine engineered anti-TENB2 antibody, or antibody drug conjugatethereof, in the preparation of a medicament useful for (i) thetherapeutic treatment or diagnostic detection of a cancer or tumor, or(ii) the therapeutic treatment or prevention of a cell proliferativedisorder.

Another embodiment of the present invention is directed to a method forinhibiting the growth of a cancer cell, wherein the growth of saidcancer cell is at least in part dependent upon the growth potentiatingeffect(s) of a TENB2 polypeptide, wherein the method comprisescontacting the TENB2 polypeptide with a cysteine engineered anti-TENB2antibody, or antibody drug conjugate thereof, thereby antagonizing thegrowth-potentiating activity of the TENB2 polypeptide and, in turn,inhibiting the growth of the cancer cell, whereby the growth of thecancer cell is inhibited.

Another embodiment of the present invention is directed to a method oftherapeutically treating a tumor in a mammal, wherein the growth of saidtumor is at least in part dependent upon the growth potentiatingeffect(s) of a TENB2 polypeptide, wherein the method comprisesadministering to the mammal a therapeutically effective amount of ananti-TENB2 cysteine engineered antibody, or antibody drug conjugatethereof, that binds to the TENB2 polypeptide, thereby antagonizing thegrowth potentiating activity of said TENB2 polypeptide and resulting inthe effective therapeutic treatment of the tumor.

The antibodies, antibody fragments, and conjugates thereof recognizeextracellular epitopes of plasma membrane TENB2 proteins that arereleased into the extracellular fluid. The invention further providesmethods for the detection, monitoring and treatment of malignancies suchas breast cancer and ovarian cancer using the antibodies, antibodyfragments and conjugates.

Antibody-drug conjugates (ADC) of the present invention may be used totreat various diseases or disorders, e.g. characterized by theoverexpression of a TENB2 tumor antigen. Exemplary conditions orhyperproliferative disorders include benign or malignant tumorsincluding prostate cancer.

The ADC compounds which are identified in the animal models andcell-based assays can be further tested in tumor-bearing higher primatesand human clinical trials. The clinical trial may be designed toevaluate the efficacy of an ADC in combinations with known therapeuticregimens, such as radiation and/or chemotherapy involving knownchemotherapeutic and/or cytotoxic agents.

Generally, the disease or disorder to be treated is a hyperproliferativedisease such as cancer. Examples of cancer to be treated herein include,but are not limited to, carcinoma, lymphoma, blastoma, sarcoma, andleukemia or lymphoid malignancies. More particular examples of suchcancers include squamous cell cancer (e.g. epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung and squamous carcinoma of thelung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer, pancreatic cancer,glioblastoma, cervical cancer, ovarian cancer, liver cancer, bladdercancer, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, as well as head andneck cancer.

For the prevention or treatment of disease, the appropriate dosage of anADC will depend on the type of disease to be treated, as defined above,the severity and course of the disease, whether the molecule isadministered for preventive or therapeutic purposes, previous therapy,the patient's clinical history and response to the antibody, and thediscretion of the attending physician. The molecule is suitablyadministered to the patient at one time or over a series of treatments.Depending on the type and severity of the disease, about 1 pg/kg to 15mg/kg (e.g. 0.1-20 mg/kg) of molecule is an initial candidate dosage foradministration to the patient, whether, for example, by one or moreseparate administrations, or by continuous infusion. A typical dailydosage might range from about 1 pg/kg to 100 mg/kg or more, depending onthe factors mentioned above. An exemplary dosage of ADC to beadministered to a patient is in the range of about 0.1 to about 10 mg/kgof patient weight.

For repeated administrations over several days or longer, depending onthe condition, the treatment is sustained until a desired suppression ofdisease symptoms occurs. An exemplary dosing regimen comprisesadministering an initial loading dose of about 4 mg/kg, followed by aweekly maintenance dose of about 2 mg/kg of an anti-TENB2 antibody.Other dosage regimens may be useful. The progress of this therapy iseasily monitored by conventional techniques and assays includingultrasound imaging.

Administration of Antibody-Drug Conjugates

The antibody-drug conjugates (ADC) of the invention may be administeredby any route appropriate to the condition to be treated. The ADC willtypically be administered parenterally, i.e. infusion, subcutaneous,intraperitoneal, intramuscular, intravenous, intradermal, intrathecaland epidural.

Pharmaceutical Formulations

Pharmaceutical formulations of therapeutic antibody-drug conjugates(ADC) of the invention are typically prepared for parenteraladministration, i.e. bolus, intravenous, intratumor injection with apharmaceutically acceptable parenteral vehicle and in a unit dosage,sterile injectable form. An antibody-drug conjugate (ADC) having thedesired degree of purity is optionally mixed with pharmaceuticallyacceptable diluents, carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences (1980) 16th edition, Osol, A. Ed.), in the formof a lyophilized formulation or an aqueous solution.

Acceptable diluents, carriers, excipients, and stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g. Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The active pharmaceutical ingredients may also be entrapped inmicrocapsules prepared, for example, by coacervation techniques or byinterfacial polymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semi permeable matrices of solidhydrophobic polymers containing the ADC, which matrices are in the formof shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.

The formulations include those suitable for the foregoing administrationroutes. The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. Techniques and formulations generally are found in Remington'sPharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

Aqueous suspensions of the invention contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, croscarmellose, povidone, methylcellulose,hydroxypropyl methylcelluose, sodium alginate, polyvinylpyrrolidone, gumtragacanth and gum acacia, and dispersing or wetting agents such as anaturally occurring phosphatide (e.g., lecithin), a condensation productof an alkylene oxide with a fatty acid (e.g., polyoxyethylene stearate),a condensation product of ethylene oxide with a long chain aliphaticalcohol (e.g., heptadecaethyleneoxycetanol), a condensation product ofethylene oxide with a partial ester derived from a fatty acid and ahexitol anhydride (e.g., polyoxyethylene sorbitan monooleate). Theaqueous suspension may also contain one or more preservatives such asethyl or n-propyl p-hydroxy-benzoate, one or more coloring agents, oneor more flavoring agents and one or more sweetening agents, such assucrose or saccharin.

The pharmaceutical compositions of ADC may be in the form of a sterileinjectable preparation, such as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according tothe known art using those suitable dispersing or wetting agents andsuspending agents which have been mentioned above. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally acceptable diluent or solvent,such as a solution in 1,3-butane-diol or prepared as a lyophilizedpowder. Among the acceptable vehicles and solvents that may be employedare water, Ringer's solution and isotonic sodium chloride solution. Inaddition, sterile fixed oils may conventionally be employed as a solventor suspending medium. For this purpose any bland fixed oil may beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid may likewise be used in the preparation ofinjectables.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, anaqueous solution intended for intravenous infusion may contain fromabout 3 to 500 μg of the active ingredient per milliliter of solution inorder that infusion of a suitable volume at a rate of about 30 mL/hr canoccur.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

Although oral administration of protein therapeutics are disfavored dueto hydrolysis or denaturation in the gut, formulations of ADC suitablefor oral administration may be prepared as discrete units such ascapsules, cachets or tablets each containing a predetermined amount ofthe ADC.

The formulations may be packaged in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water, for injection immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Preferred unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

The compositions of the invention may also be formulated asimmunoliposomes. A “liposome” is a small vesicle composed of varioustypes of lipids, phospholipids and/or surfactant which is useful fordelivery of a drug to a mammal. The components of the liposome arecommonly arranged in a bilayer formation, similar to the lipidarrangement of biological membranes. Liposomes containing the antibodyare prepared by methods known in the art, such as described in Epsteinet al (1985) Proc. Natl. Acad. Sci. USA 82:3688; Hwang et al (1980)Proc. Natl Acad. Sci. USA 77:4030; U.S. Pat. No. 4,485,045; U.S. Pat.No. 4,544,545; U.S. Pat. No. 5,013,556; WO 97/38731. Liposomes can begenerated by the reverse phase evaporation method with a lipidcomposition comprising phosphatidylcholine, cholesterol andPEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes may beextruded through filters of defined pore size to yield liposomes withthe desired diameter. Fab′fragments of the compositions of the presentinvention can be conjugated to liposomes (Martin et al (1982) J. Biol.Chem. 257:286-288), via a disulfide interchange reaction. Achemotherapeutic agent is optionally contained within the liposome(Gabizon et al (1989) J. National Cancer Inst. 81(19): 1484.

Combination Therapy

An antibody-drug conjugate (ADC) of the invention may be combined in apharmaceutical combination formulation, or dosing regimen as combinationtherapy, with a second compound having anti-cancer properties. Thesecond compound of the pharmaceutical combination formulation or dosingregimen preferably has complementary activities to the ADC of thecombination such that they do not adversely affect each other.

The second compound may be a chemotherapeutic agent, cytotoxic agent,cytokine, growth inhibitory agent, anti-hormonal agent, and/orcardioprotectant. Such molecules are suitably present in combination inamounts that are effective for the purpose intended. A pharmaceuticalcomposition containing an ADC of the invention may also have atherapeutically effective amount of a chemotherapeutic agent such as atubulin-forming inhibitor, a topoisomerase inhibitor, a DNAintercalator, or a DNA binder.

Other therapeutic regimens may be combined with the administration of ananticancer agent identified in accordance with this invention. Thecombination therapy may be administered as a simultaneous or sequentialregimen. When administered sequentially, the combination may beadministered in two or more administrations. The combined administrationincludes coadministration, using separate formulations or a singlepharmaceutical formulation, and consecutive administration in eitherorder, wherein preferably there is a time period while both (or all)active agents simultaneously exert their biological activities.

In one embodiment, treatment with an ADC involves the combinedadministration of a cysteine engineered anti-TENB2 antibody orantibody-drug conjugate thereof, and one or more chemotherapeuticagents, therapeutic biological, or growth inhibitory agents, includingcoadministration of cocktails of different chemotherapeutic agents.Chemotherapeutic agents include, but are not limited to: taxanes (suchas paclitaxel and docetaxel); platinum-containing compounds, such ascarboplatin; EGFR inhibitors such as erlotinib, and gefitinib; tyrosinekinase inhibitors such as imatinib; and anthracycline antibiotics (suchas doxorubicin or doxil). Therapeutic biological agents to be used incombination with a cysteine engineered anti-TENB2 antibody orantibody-drug conjugate thereof include bevacizumab (Avastin®) orpertuzumab (Omnitarg™, Genentech Inc). Preparation and dosing schedulesfor such chemotherapeutic agents may be used according to manufacturer'sinstructions or as determined empirically by the skilled practitioner.

Preparation and dosing schedules for such chemotherapy are alsodescribed in “Chemotherapy Service”, (1992) Ed., M. C. Perry, Williams &Wilkins, Baltimore, Md.

The ADC may be combined with an anti-hormonal compound; e.g., ananti-estrogen compound such as tamoxifen; an anti-progesterone such asonapristone (EP 616812); or an anti-androgen such as flutamide, indosages known for such molecules. Where the cancer to be treated ishormone independent cancer, the patient may previously have beensubjected to anti-hormonal therapy and, after the cancer becomes hormoneindependent, the ADC (and optionally other agents as described herein)may be administered to the patient. It may be beneficial to alsocoadminister a cardioprotectant (to prevent or reduce myocardialdysfunction associated with the therapy) or one or more cytokines to thepatient. In addition to the above therapeutic regimes, the patient maybe subjected to surgical removal of cancer cells and/or radiationtherapy.

Suitable dosages for any of the above coadministered agents are thosepresently used and may be lowered due to the combined action (synergy)of the newly identified agent and other chemotherapeutic agents ortreatments.

The combination therapy may provide “synergy” and prove “synergistic”,i.e. the effect achieved when the active ingredients used together isgreater than the sum of the effects that results from using thecompounds separately. A synergistic effect may be attained when theactive ingredients are: (1) co-formulated and administered or deliveredsimultaneously in a combined, unit dosage formulation; (2) delivered byalternation or in parallel as separate formulations; or (3) by someother regimen. When delivered in alternation therapy, a synergisticeffect may be attained when the compounds are administered or deliveredsequentially, e.g. by different injections in separate syringes. Ingeneral, during alternation therapy, an effective dosage of each activeingredient is administered sequentially, i.e. serially, whereas incombination therapy, effective dosages of two or more active ingredientsare administered together.

Metabolites of the Antibody-Drug Conjugates

Also falling within the scope of this invention are the in vivometabolic products of the ADC compounds described herein, to the extentsuch products are novel and unobvious over the prior art. Such productsmay result for example from the oxidation, reduction, hydrolysis,amidation, esterification, enzymatic cleavage, and the like, of theadministered compound. Accordingly, the invention includes novel andunobvious compounds produced by a process comprising contacting acompound of this invention with a mammal for a period of time sufficientto yield a metabolic product thereof.

Metabolite products typically are identified by preparing aradiolabelled (e.g. ¹⁴C or ³H) ADC, administering it parenterally in adetectable dose (e.g. greater than about 0.5 mg/kg) to an animal such asrat, mouse, guinea pig, monkey, or to man, allowing sufficient time formetabolism to occur (typically about 30 seconds to 30 hours) andisolating its conversion products from the urine, blood or otherbiological samples. These products are easily isolated since they arelabeled (others are isolated by the use of antibodies capable of bindingepitopes surviving in the metabolite). The metabolite structures aredetermined in conventional fashion, e.g. by MS, LC/MS or NMR analysis.In general, analysis of metabolites is done in the same way asconventional drug metabolism studies well-known to those skilled in theart. The conversion products, so long as they are not otherwise found invivo, are useful in diagnostic assays for therapeutic dosing of the ADCcompounds of the invention.

Articles of Manufacture

In another embodiment of the invention, an article of manufacture, or“kit”, containing materials useful for the treatment of the disordersdescribed above is provided. The article of manufacture comprises acontainer and a label or package insert on or associated with thecontainer. The package insert may refer to instructions customarilyincluded in commercial packages of therapeutic products and that containinformation about the indications, usage, dosage, administration,contraindications and/or warnings concerning the use of such therapeuticproducts. Suitable containers include, for example, bottles, vials,syringes, blister pack, etc. The containers may be formed from a varietyof materials such as glass or plastic.

In one embodiment, the article of manufacture comprises a container anda formulation of a cysteine engineered anti-TENB2 antibody, orantibody-drug conjugate thereof, contained within the container. Thearticle may further optionally comprise a label affixed to thecontainer, or a package insert included with the container, that refersto the use of the composition of matter for the therapeutic treatment ordiagnostic detection of a tumor. The container holding the formulationis effective for storing and delivering the therapeutic and may have asterile access port (for example the container may be an intravenoussolution bag or a vial having a stopper pierceable by a hypodermicinjection needle). The label or package insert indicates that theformulation is used for treating the condition of choice, such ascancer. Alternatively, or additionally, the article of manufacture mayfurther comprise a second (or third) container comprising apharmaceutically-acceptable buffer, such as bacteriostatic water forinjection (BWFI), phosphate-buffered saline, Ringer's solution anddextrose solution. It may further include other materials desirable froma commercial and user standpoint, including other buffers, diluents,filters, needles, and syringes.

The following examples are offered for illustrative purposes only, andare not intended to limit the scope of the present invention in any way.

All patent and literature references cited in the present specificationare hereby incorporated by reference in their entirety.

EXAMPLES

Commercially available reagents referred to in the examples were usedaccording to manufacturer's instructions unless otherwise indicated. Thesource of those cells identified in the following examples, andthroughout the specification, by ATCC accession numbers is the AmericanType Culture Collection, Manassas, Va.

Example 1 Preparation of Anti-TMEFF2#19 Antibodies

Humanized TMEFF2#19 antibodies were prepared according to PCT/US03/07209(U.S. Pat. No. 7,288,248). FIG. 1 shows the heavy chain amino acidsequence (SEQ ID NO:1) and the light chain amino acid sequence (SEQ IDNO:2).

Example 2 Preparation of Cysteine Engineered Anti-TENB2 Antibodies forConjugation by Reduction and Reoxidation

Full length, cysteine engineered anti-TENB2 monoclonal antibodies(ThioMabs) expressed in CHO cells bear cysteine adducts (cystines) onthe engineered cysteines due to cell culture conditions. To liberate thereactive thiol groups of the engineered cysteines, the ThioMabs aredissolved in 500 mM sodium borate and 500 mM sodium chloride at about pH8.0 and reduced with about a 50-100 fold excess of 1 mM TCEP(tris(2-carboxyethyl)phosphine hydrochloride; Getz et al (1999) Anal.Biochem. Vol 273:73-80; Soltec Ventures, Beverly, Mass.) for about 1-2hrs at 37° C. The reduced ThioMab (FIG. 6) is diluted and loaded onto aHiTrap S column in 10 mM sodium acetate, pH 5, and eluted with PBScontaining 0.3M sodium chloride. The eluted reduced ThioMab is treatedwith 2 mM dehydroascorbic acid (dhAA) at pH 7 for 3 hours, or 2 mMaqueous copper sulfate (CuSO₄) at room temperature overnight. Ambientair oxidation may also be effective. The buffer is exchanged by elutionover Sephadex G25 resin and eluted with PBS with 1 mM DTPA. The thiol/Abvalue is checked by determining the reduced antibody concentration fromthe absorbance at 280 nm of the solution and the thiol concentration byreaction with DTNB (Aldrich, Milwaukee, Wis.) and determination of theabsorbance at 412 nm.

Example 3 Conjugation of Cysteine Engineered Anti-TENB2 Antibodies andDrug-Linker Intermediates

After the reduction and reoxidation procedures of Example 2, thecysteine engineered anti-TENB2 antibody is dissolved in PBS (phosphatebuffered saline) buffer and chilled on ice. About 1.5 molar equivalentsrelative to engineered cysteines per antibody of an auristatin druglinker intermediate, such as MC-MMAE (maleimidocaproyl-monomethylauristatin E), MC-MMAF, MC-val-cit-PAB-MMAE, or MC-val-cit-PAB-MMAF,with a thiol-reactive functional group such as maleimido, is dissolvedin DMSO, diluted in acetonitrile and water, and added to the chilledreduced, reoxidized antibody in PBS. After about one hour, an excess ofmaleimide is added to quench the reaction and cap any unreacted antibodythiol groups. The reaction mixture is concentrated by centrifugalultrafiltration and the cysteine engineered anti-TENB2 antibody drugconjugate is purified and desalted by elution through G25 resin in PBS,filtered through 0.2 m filters under sterile conditions, and frozen forstorage.

By the procedure above, the following cysteine engineered anti-TENB2antibody drug conjugates were prepared:

thio hu TMEFF2#19-MC-MMAF by conjugation of A114C (Kabat) thio huTMEFF2#19 and MC-MMAF; and

thio hu TMEFF2#19-MC-val-cit-PAB-MMAE by conjugation of A114C (Kabat)thio hu TMEFF2#19 and MC-val-cit-PAB-MMAE.

Example 4 Materials and Methods for IHC, Internalization Studies, FACS,Cell Killing Assays, Western Blots, Xenograft Studies, PharmacokineticStudies and Safety Assessments

Antibodies and Recombinant Proteins: Humanized anti-tenb2 Mab PR1 wasobtained from PDL. ThioMab anti-tenB2 PR1(HC-A121C; sequentialnumbering) and tenB2ECD Flag protein were produced as discussed above.

Cell Lines and Human Tumor Xenografts: PC3 is a human prostate carcinomacell line (ATCC). PC3TenB2 Medium stable cell line was generated byGenentech. Human prostate explant models, LuCap70, 77 and 96.1 wereobtained from the University of Washington.

RNA and Protein Expression: RNA expression analysis, Immunologicalprocedures (IHC, Western), antibody binding (FACS) and internalizationfollowed previously published methods (Cancer research 64, 781-788(2004)).

Preparation of Conventional or ThioMabAnti-TenB2-Valine-Citrulline(vc)-Monomethylauristatine E(MMAE) andMC-MMAF Armed Drug Conjugated(ADC): The conjugation of conventional,thio-mab and control mab with vc-MMAE, MC-MMAF ADC was performed asdescribed above.

In vitro Cell Killing and in vivo Studies: The cell killing assay wasdone similar to as described in Cancer research 64, 781-788 (2004). Eachprostate explant model tumor cell line was maintained by serialtransplantation in castrated (androgen independent model, LuCap70) oruncastrated (androgen dependent model, LuCAP77 and LuCAP96. 1), maleSCID-beige mice from Charles River lab. Tumors were measured once totwice per week for duration of the study.

Rats and Cynomolgus Monkeys Models for Safety Assessment: Anti-tenb2 Mabspecifically recognized human, monkey and rats tenb2 target (FS Idomain).

Pharmacokinetic Study: Standard protocol and assay methods were used.

The data demonstrate that human TenB2 (TMEFF2) is generally restrictedto expression in the prostate and CNS, with significantly elevatedlevels in cancerous prostate. Anti-TenB2 antibodies were alsodemonstrated to be rapidly internalized. These antibodies, whenconjugated to MMAE and MMAF were shown to kill prostate tumor cells invitro and in vivo in various cell killing assays. Furthermore,efficacious doses of TENB2-ADCs were significantly lower than those thatare required to elicit toxic effects in rodents and primates.

The foregoing written specification is considered to be sufficient toenable one skilled in the art to practice the invention. The presentinvention is not to be limited in scope by the construct deposited,since the deposited embodiment is intended as a single illustration ofcertain aspects of the invention and any constructs that arefunctionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. A cysteine engineered anti-TENB2 antibody comprising one or more freecysteine amino acids and a sequence selected from SEQ ID NOS:8-23. 2.The cysteine engineered anti-TENB2 antibody of claim 1 wherein thecysteine engineered anti-TENB2 antibody binds to a TENB2 polypeptide. 3.The cysteine engineered anti-TENB2 antibody of claim 1 prepared by aprocess comprising replacing one or more amino acid residues of a parentanti-TENB2 antibody by cysteine. 4-7. (canceled)
 8. The cysteineengineered anti-TENB2 antibody of claim 1 comprising a heavy chainsequence comprising: SEQ ID NO: 3MAVLGLLLCLVTFPSCVLSDVQLQESGPGLVKPSETLSLTCAVSGYSITSGYYWSWIRQPPGKGLEWMGFISYDGSNKYNPSLKNRITISRDTSKNQFSLKLSSVTAADTAVYYCARGLRRGDYSMDYWGQGTLVTVSSCSTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK


9. The cysteine engineered anti-TENB2 antibody of claim 1 comprising alight chain sequence comprising: SEQ ID NO: 2MDFQVQIFSFLLISASVIMSRGDIQMTQSPSSLSASVGDRVTITCKASQNVVTAVAWYQQKPGKAPKLLIYESASNRHTGVPSRFSGSGSGTDFTLTISSLQPEDGATYYCQQYSSYPFTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTK SFNRGEC


10. The cysteine engineered anti-TENB2 antibody of claim 1 wherein theparent anti-TENB2 antibody is selected from a monoclonal antibody, abispecific antibody, a chimeric antibody, a human antibody, and ahumanized antibody.
 11. The cysteine engineered anti-TENB2 antibody ofclaim 1 which is an antibody fragment. 12-15. (canceled)
 16. A method ofdetermining the presence of a TENB2 protein in a sample suspected ofcontaining said protein, said method comprising exposing said sample toa cysteine engineered anti-TENB2 antibody of claim 1 and determiningbinding of said antibody to said TENB2 protein in said sample, whereinbinding of the antibody to said protein is indicative of the presence ofsaid protein in said sample.
 17. The method of claim 16 wherein saidsample comprises a cell suspected of expressing said TENB2 protein. 18.The method of claim 16 wherein said cell is a prostate, ovarian, breast,lung, or pancreatic cancer cell.
 19. The method of claim 16 wherein theantibody is covalently attached to a label selected from a fluorescentdye, a radioisotope, biotin, or a metal-complexing ligand.
 20. Apharmaceutical formulation comprising the cysteine engineered anti-TENB2antibody of claim 1, and a pharmaceutically acceptable diluent, carrieror excipient.
 21. The cysteine engineered anti-TENB2 antibody of claim 1wherein the antibody is covalently attached to an auristatin drug moietywhereby an antibody drug conjugate is formed.
 22. The antibody-drugconjugate of claim 21 comprising a cysteine engineered anti-TENB2antibody (Ab), and an auristatin drug moiety (D) wherein the cysteineengineered anti-TENB2 antibody is attached through one or more freecysteine amino acids by a linker moiety (L) to D; the compound havingFormula I:Ab-(L-D)_(p)  I where p is 1, 2, 3, or
 4. 23-33. (canceled)
 34. Theantibody-drug conjugate compound of claim 21 wherein the parentanti-TENB2 antibody is selected from a monoclonal antibody, a bispecificantibody, a chimeric antibody, a human antibody, and a humanizedantibody.
 35. The antibody-drug conjugate compound of claim 21 whereinthe parent anti-TENB2 antibody is an antibody fragment.
 36. (canceled)37. The antibody drug conjugate of claim 21 wherein the auristatin isMMAE or MMAF.
 38. The antibody drug conjugate of claim 21 wherein L isMC-val-cit-PAB or MC.
 39. The antibody drug conjugate of claim 21wherein L is SMCC, SPP, or BMPEO.
 40. An antibody-drug conjugatecompound selected from the structures:

wherein Val is valine; Cit is citrulline; p is 1, 2, 3, or 4; and Ab isa cysteine engineered anti-TENB2 antibody of claim
 1. 41. The antibodydrug conjugate of claim 40 wherein Ab comprises SEQ ID NO:1.
 42. Theantibody drug conjugate of claim 40 wherein Ab comprises SEQ ID NO:2.43. The antibody drug conjugate of claim 40 wherein Ab comprises SEQ IDNO: 1 and SEQ ID NO:2.
 44. An assay for detecting cancer cellscomprising: (a) exposing cells to an antibody-drug conjugate compound ofclaim 21; and (b) determining the extent of binding of the antibody-drugconjugate compound to the cells.
 45. The assay of claim 44 wherein thecells are prostate, pancreatic, lung, breast, colon or ovarian tumorcells.
 46. A method of inhibiting cellular proliferation comprisingtreating mammalian tumor cells in a cell culture medium with anantibody-drug conjugate compound of claim 21, whereby proliferation ofthe tumor cells is inhibited.
 47. The method of claim 46 wherein themammalian tumor cells are ovarian tumor cells.
 48. A pharmaceuticalformulation comprising the antibody drug conjugate of claim 21, and apharmaceutically acceptable diluent, carrier or excipient.
 49. Thepharmaceutical formulation of claim 48 further comprising atherapeutically effective amount of a chemotherapeutic agent selectedfrom letrozole, oxaliplatin, doxetaxel, 5-FU, lapatinib, capecitabine,leucovorin, erlotinib, pertuzumab, bevacizumab, and gemcitabine.
 50. Amethod of treating cancer comprising administering to a patient thepharmaceutical formulation of claim
 48. 51. The method of claim 50wherein the cancer is selected from the group consisting of prostatecancer, cancer of the urinary tract, pancreatic cancer, lung cancer,breast cancer, colon cancer and ovarian cancer.
 52. The method of claim50 wherein the patient is administered a chemotherapeutic agent incombination with the antibody-drug conjugate compound, where thechemotherapeutic agent is selected from letrozole, cisplatin,carboplatin, taxol, paclitaxel, oxaliplatin, doxetaxel, 5-FU,leucovorin, erlotinib, pertuzumab, bevacizumab, lapatinib, andgemcitabine.
 53. An article of manufacture comprising the pharmaceuticalformulation of claim 48; a container; and a package insert or labelindicating that the compound can be used to treat cancer characterizedby the overexpression of a TENB2 polypeptide.
 54. The article ofmanufacture of claim 53 wherein the cancer is ovarian cancer, prostatecancer, cancer of the urinary tract, pancreatic cancer, lung cancer,breast cancer, or colon cancer.
 55. A method for making an antibody drugconjugate compound comprising a cysteine engineered anti-TENB2 antibody(Ab) of claim 1, and an auristatin drug moiety (D) wherein the cysteineengineered antibody is attached through the one or more engineeredcysteine amino acids by a linker moiety (L) to D; the compound havingFormula I:Ab-(L-D)_(p)  I where p is 1, 2, 3, or 4; the method comprising thesteps of: (a) reacting an engineered cysteine group of the cysteineengineered antibody with a linker reagent to form antibody-linkerintermediate Ab-L; and (b) reacting Ab-L with an activated drug moietyD; whereby the antibody-drug conjugate is formed; or comprising thesteps of: (c) reacting a nucleophilic group of a drug moiety with alinker reagent to form drug-linker intermediate D-L; and (d) reactingD-L with an engineered cysteine group of the cysteine engineeredantibody; whereby the antibody-drug conjugate is formed.
 56. The methodof claim 55 further comprising the step of expressing the cysteineengineered antibody in chinese hamster ovary (CHO) cells.
 57. The methodof claim 56 further comprising the step of treating the expressedcysteine engineered antibody with a reducing agent.
 58. The method ofclaim 57 wherein the reducing agent is selected from TCEP and DTT. 59.The method of claim 57 further comprising the step of treating theexpressed cysteine engineered antibody with an oxidizing agent, aftertreating with the reducing agent.
 60. The method of claim 59 wherein theoxidizing agent is selected from copper sulfate, dehydroascorbic acid,and air.